Insecticidal proteins and methods for their use

ABSTRACT

Compositions and methods for controlling pests are provided. The methods involve transforming organisms with a nucleic acid sequence encoding an insecticidal protein. In particular, the nucleic acid sequences are useful for preparing plants and microorganisms that possess insecticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. Compositions are insecticidal nucleic acids and proteins of bacterial species. The sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest including plants, as probes for the isolation of other homologous (or partially homologous) genes. The pesticidal proteins find use in controlling, inhibiting growth or killing Lepidopteran, Coleopteran, Dipteran, fungal, Hemipteran and nematode pest populations and for producing compositions with insecticidal activity.

GOVERNMENT SUPPORT

The government has certain rights in the invention pursuant to AgreementNo. LB09005376.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

A sequence listing having the file name “5914-PCT_seq_list.txt” createdon Nov. 19, 2015, and having a size of 542 kilobytes is filed incomputer readable form concurrently with the specification. The sequencelisting is part of the specification and is herein incorporated byreference in its entirety.

FIELD

This disclosure relates to the field of molecular biology. Provided arenovel genes that encode pesticidal proteins. These pesticidal proteinsand the nucleic acid sequences that encode them are useful in preparingpesticidal formulations and in the production of transgenicpest-resistant plants.

BACKGROUND

Biological control of insect pests of agricultural significance using amicrobial agent, such as fungi, bacteria or another species of insectaffords an environmentally friendly and commercially attractivealternative to synthetic chemical pesticides. Generally speaking, theuse of biopesticides presents a lower risk of pollution andenvironmental hazards and biopesticides provide greater targetspecificity than is characteristic of traditional broad-spectrumchemical insecticides. In addition, biopesticides often cost less toproduce and thus improve economic yield for a wide variety of crops.

Certain species of microorganisms of the genus Bacillus are known topossess pesticidal activity against a range of insect pests includingLepidoptera, Diptera, Coleoptera, Hemiptera and others. Bacillusthuringiensis (Bt) and Bacillus popilliae are among the most successfulbiocontrol agents discovered to date. Insect pathogenicity has also beenattributed to strains of B. larvae, B. lentimorbus, B. sphaericus and B.cereus. Microbial insecticides, particularly those obtained fromBacillus strains, have played an important role in agriculture asalternatives to chemical pest control.

Crop plants have been developed with enhanced insect resistance bygenetically engineering crop plants to produce pesticidal proteins fromBacillus. For example, corn and cotton plants have been geneticallyengineered to produce pesticidal proteins isolated from strains of Bt.These genetically engineered crops are now widely used in agricultureand have provided the farmer with an environmentally friendlyalternative to traditional insect-control methods. While they haveproven to be very successful commercially, these genetically engineered,insect-resistant crop plants provide resistance to only a narrow rangeof the economically important insect pests. In some cases, insects candevelop resistance to different insecticidal compounds, which raises theneed to identify alternative biological control agents for pest control.

Accordingly, there remains a need for new pesticidal proteins withdifferent ranges of insecticidal activity against insect pests, e.g.,insecticidal proteins which are active against a variety of insects inthe order Lepidoptera and the order Coleoptera including but not limitedto insect pests that have developed resistance to existing insecticides.

SUMMARY

Compositions and methods for conferring pesticidal activity to bacteria,plants, plant cells, tissues and seeds are provided. Compositionsinclude nucleic acid molecules encoding sequences for pesticidal andinsecticidal polypeptides, vectors comprising those nucleic acidmolecules, and host cells comprising the vectors. Compositions alsoinclude the pesticidal polypeptide sequences and antibodies to thosepolypeptides. The nucleic acid sequences can be used in DNA constructsor expression cassettes for transformation and expression in organisms,including microorganisms and plants. The nucleotide or amino acidsequences may be synthetic sequences that have been designed forexpression in an organism including, but not limited to, a microorganismor a plant. Compositions also comprise transformed bacteria, plants,plant cells, tissues and seeds.

In particular, isolated or recombinant nucleic acid molecules areprovided encoding Pseudomonas Insecticidal Protein-45-1 (PIP-45-1)polypeptides including amino acid substitutions, deletions, insertions,and fragments thereof, and combinations thereof. Additionally, aminoacid sequences corresponding to the PIP-45-1 polypeptides areencompassed. Provided are isolated or recombinant nucleic acid moleculescapable of encoding a PIP-45-1 polypeptide of SEQ ID NO: 1 as well asamino acid substitutions, deletions, insertions, fragments thereof andcombinations thereof. Nucleic acid sequences that are complementary to anucleic acid sequence of the embodiments or that hybridize to a sequenceof the embodiments are also encompassed. Also provided are isolated orrecombinant PIP-45-1 polypeptides of SEQ ID NO: 1 as well as amino acidsubstitutions, deletions, insertions, fragments thereof and combinationsthereof.

In particular, isolated or recombinant nucleic acid molecules areprovided encoding Pseudomonas Insecticidal Protein-45-2 (PIP-45-2)polypeptides including amino acid substitutions, deletions, insertions,and fragments thereof, and combinations thereof. Additionally, aminoacid sequences corresponding to the PIP-45-2 polypeptides areencompassed. Provided are isolated or recombinant nucleic acid moleculescapable of encoding a PIP-45-2 polypeptide of SEQ ID NO: 2 as well asamino acid substitutions, deletions, insertions, fragments thereof andcombinations thereof. Nucleic acid sequences that are complementary to anucleic acid sequence of the embodiments or that hybridize to a sequenceof the embodiments are also encompassed. Also provided are isolated orrecombinant PIP-45-2 polypeptides of SEQ ID NO: 2 as well as amino acidsubstitutions, deletions, insertions, fragments thereof and combinationsthereof.

In particular, isolated or recombinant nucleic acid molecules areprovided encoding Pseudomonas Insecticidal Protein-64-1 (PIP-64-1)polypeptides including amino acid substitutions, deletions, insertions,and fragments thereof, and combinations thereof. Additionally, aminoacid sequences corresponding to the PIP-64-1 polypeptides areencompassed. Provided are isolated or recombinant nucleic acid moleculescapable of encoding a PIP-64-1 polypeptide of SEQ ID NO: 53 as well asamino acid substitutions, deletions, insertions, fragments thereof andcombinations thereof. Nucleic acid sequences that are complementary to anucleic acid sequence of the embodiments or that hybridize to a sequenceof the embodiments are also encompassed. Also provided are isolated orrecombinant PIP-64-1 polypeptides of SEQ ID NO: 53 as well as amino acidsubstitutions, deletions, insertions, fragments thereof and combinationsthereof.

In particular, isolated or recombinant nucleic acid molecules areprovided encoding Pseudomonas Insecticidal Protein-64-2 (PIP-64-2)polypeptides including amino acid substitutions, deletions, insertions,and fragments thereof, and combinations thereof. Additionally, aminoacid sequences corresponding to the PIP-64-2 polypeptides areencompassed. Provided are isolated or recombinant nucleic acid moleculescapable of encoding a PIP-64-2 polypeptide of SEQ ID NO: 54 as well asamino acid substitutions, deletions, insertions, fragments thereof andcombinations thereof. Nucleic acid sequences that are complementary to anucleic acid sequence of the embodiments or that hybridize to a sequenceof the embodiments are also encompassed. Also provided are isolated orrecombinant PIP-64-2 polypeptides of SEQ ID NO: 54 as well as amino acidsubstitutions, deletions, insertions, fragments thereof and combinationsthereof.

In particular, isolated or recombinant nucleic acid molecules areprovided encoding Pseudomonas Insecticidal Protein-74-1 (PIP-74-1)polypeptides including amino acid substitutions, deletions, insertions,and fragments thereof, and combinations thereof. Additionally, aminoacid sequences corresponding to the PIP-74-1 polypeptides areencompassed. Provided are isolated or recombinant nucleic acid moleculescapable of encoding a PIP-74-1 polypeptide of SEQ ID NO: 73 as well asamino acid substitutions, deletions, insertions, fragments thereof andcombinations thereof. Nucleic acid sequences that are complementary to anucleic acid sequence of the embodiments or that hybridize to a sequenceof the embodiments are also encompassed. Also provided are isolated orrecombinant PIP-74-1 polypeptides of SEQ ID NO: 73 as well as amino acidsubstitutions, deletions, insertions, fragments thereof and combinationsthereof.

In particular, isolated or recombinant nucleic acid molecules areprovided encoding Pseudomonas Insecticidal Protein-74-2 (PIP-74-2)polypeptides including amino acid substitutions, deletions, insertions,and fragments thereof, and combinations thereof. Additionally, aminoacid sequences corresponding to the PIP-74-2 polypeptides areencompassed. Provided are isolated or recombinant nucleic acid moleculescapable of encoding a PIP-74-2 polypeptide of SEQ ID NO: 74 as well asamino acid substitutions, deletions, insertions, fragments thereof andcombinations thereof. Nucleic acid sequences that are complementary to anucleic acid sequence of the embodiments or that hybridize to a sequenceof the embodiments are also encompassed. Also provided are isolated orrecombinant PIP-74-2 polypeptides of SEQ ID NO: 74 as well as amino acidsubstitutions, deletions, insertions, fragments thereof and combinationsthereof.

In particular, isolated or recombinant nucleic acid molecules areprovided encoding Pseudomonas Insecticidal Protein-75 (PIP-75)polypeptides including amino acid substitutions, deletions, insertions,and fragments thereof, and combinations thereof. Additionally, aminoacid sequences corresponding to the PIP-75 polypeptides are encompassed.Provided are isolated or recombinant nucleic acid molecules capable ofencoding a PIP-75 polypeptide of SEQ ID NO: 79 as well as amino acidsubstitutions, deletions, insertions, fragments thereof and combinationsthereof. Nucleic acid sequences that are complementary to a nucleic acidsequence of the embodiments or that hybridize to a sequence of theembodiments are also encompassed. Also provided are isolated orrecombinant PIP-75 polypeptides of SEQ ID NO: 79 as well as amino acidsubstitutions, deletions, insertions, fragments thereof and combinationsthereof.

In particular, isolated or recombinant nucleic acid molecules areprovided encoding Pseudomonas Insecticidal Protein-77 (PIP-77)polypeptides including amino acid substitutions, deletions, insertions,and fragments thereof, and combinations thereof.

Additionally, amino acid sequences corresponding to the PIP-77polypeptides are encompassed. Provided are isolated or recombinantnucleic acid molecules capable of encoding a PIP-77 polypeptide of SEQID NO: 88 as well as amino acid substitutions, deletions, insertions,fragments thereof and combinations thereof. Nucleic acid sequences thatare complementary to a nucleic acid sequence of the embodiments or thathybridize to a sequence of the embodiments are also encompassed. Alsoprovided are isolated or recombinant PIP-77 polypeptides of SEQ ID NO:88 as well as amino acid substitutions, deletions, insertions, fragmentsthereof and combinations thereof.

Methods are provided for producing the insecticidal polypeptides and forusing these polypeptides for controlling or killing a Lepidopteran,Coleopteran, nematode, fungi, and/or Dipteran pests. The transgenicplants of the embodiments express one or more of the pesticidalsequences disclosed herein. In various embodiments, the transgenic plantfurther comprises one or more additional genes for insect resistance,for example, one or more additional genes for controlling Coleopteran,Lepidopteran, Hemipteran or nematode pests. It will be understood by oneof skill in the art that the transgenic plant may comprise any geneimparting an agronomic trait of interest.

Methods for detecting the nucleic acids and polypeptides of theembodiments in a sample are also included. A kit for detecting thepresence of an insecticidal polypeptide of the disclosure or detectingthe presence of a nucleotide sequence encoding an insecticidalpolypeptide of the disclosure in a sample is provided. The kit may beprovided along with all reagents and control samples necessary forcarrying out a method for detecting the intended agent, as well asinstructions for use.

The compositions and methods of the embodiments are useful for theproduction of organisms with enhanced pest resistance or tolerance.These organisms and compositions comprising the organisms are desirablefor agricultural purposes. The compositions of the embodiments are alsouseful for generating altered or improved proteins that have pesticidalactivity or for detecting the presence of the insecticidal polypeptidesof the disclosure or nucleic acids encoding same in products ororganisms.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1a-1m shows the amino acid sequence alignment of PIP-45Aa-1 (SEQ IDNO: 1), PIP-45Ab-1 (SEQ ID NO: 3), PIP-45Ac-1 (SEQ ID NO: 5), PIP-45Ad-1(SEQ ID NO: 7), PIP-45Ae-1 (SEQ ID NO: 9), PIP-45Af-1 (SEQ ID NO: 11),PIP-45Ba-1 (SEQ ID NO: 13), PIP-45Bb-1 (SEQ ID NO: 15), PIP-45Bc-1 (SEQID NO: 17), PIP-45Bd-1 (SEQ ID NO: 19), PIP-45Be-1 (SEQ ID NO: 21),PIP-45Bf-1 (SEQ ID NO: 23), PIP-45Bg-1 (SEQ ID NO: 25), PIP-45Bh-1 (SEQID NO: 27), PIP-45Bi-1 (SEQ ID NO: 29), PIP-45Bj-1 (SEQ ID NO: 31),PIP-45Bk-1 (SEQ ID NO: 33), PIP-45Bl-1 (SEQ ID NO: 232), PIP-45Bm-1 (SEQID NO: 234), PIP-45Ca-1 (SEQ ID NO: 35), PIP-45Cb-1 (SEQ ID NO: 37),PIP-45Cc-1 (SEQ ID NO: 39), PIP-45Cd-1 (SEQ ID NO: 41), PIP-45Ce-1 (SEQID NO: 43), PIP-45Cf-1 (SEQ ID NO: 236), PIP-45Da-1 (SEQ ID NO: 45),PIP-45Db-1 (SEQ ID NO: 47), PIP-45Ea-1 (SEQ ID NO: 49), and PIP-45Ga-1(SEQ ID NO: 51). The amino acid diversity between the PIP-45-1polypeptide homologs is indicated with shading.

FIG. 2a-2l shows an alignment of the amino acid sequences of PIP-45Aa-2(SEQ ID NO: 2), PIP-45Ab-2 (SEQ ID NO: 4), PIP-45Ac-2 (SEQ ID NO: 6),PIP-45Ad-2 (SEQ ID NO: 8), PIP-45Ae-2 (SEQ ID NO: 10), PIP-45Af-2 (SEQID NO: 12), PIP-45Ba-2 (SEQ ID NO: 14), PIP-45Bb-2 (SEQ ID NO: 16),PIP-45Bc-2 (SEQ ID NO: 18), PIP-45Bd-2 (SEQ ID NO: 20), PIP-45Be-2 (SEQID NO: 22), PIP-45Bf-2 (SEQ ID NO: 24), PIP-45Bg-2 (SEQ ID NO: 26),PIP-45Bh-2 (SEQ ID NO: 28), PIP-45Bi-2 (SEQ ID NO: 30), PIP-45Bj-2 (SEQID NO: 32), PIP-45Bk-2 (SEQ ID NO: 34), PIP-45Bl-2 (SEQ ID NO: 233),PIP-45Bm-2 (SEQ ID NO: 235), PIP-45Ca-2 (SEQ ID NO: 36), PIP-45Cb-2 (SEQID NO: 38), PIP-45Cc-2 (SEQ ID NO: 40), PIP-45Cd-2 (SEQ ID NO: 42),PIP-45Ce-2 (SEQ ID NO: 44), PIP-45Cf-2 (SEQ ID NO: 237), PIP-45Da-2 (SEQID NO: 46), PIP-45Db-2 (SEQ ID NO: 48), PIP-45Ea-2 (SEQ ID NO: 50), andPIP-45Ga-2 (SEQ ID NO: 52). The amino acid diversity between thePIP-45-2 polypeptide homologs is indicated with shading.

FIG. 3a-3b shows the amino acid sequence alignment of PIP-64Aa-1 (SEQ IDNO: 53) PIP-64Ba-1 (SEQ ID NO: 238), PIP-64Ca-1 (SEQ ID NO: 56),PIP-64Ea-1 (SEQ ID NO: 58), PIP-64Eb-1 (SEQ ID NO: 60), PIP-64Ec-1 (SEQID NO: 62), PIP-64Ga-1 (SEQ ID NO: 64), PIP-64Ha-1 (SEQ ID NO: 65),PIP-64Hb-1 (SEQ ID NO: 67), PIP-64Hc-1 (SEQ ID NO: 69), and PIP-64Hd-1(SEQ ID NO: 71). The amino acid diversity between the PIP-64-1polypeptide homologs is indicated with shading.

FIG. 4a-4b shows the amino acid sequence alignment of PIP-64Aa-2 (SEQ IDNO: 54), PIP-64Ab-2 (SEQ ID NO: 55), PIP-64Ba-2 (SEQ ID NO: 239),PIP-64Ca-2 (SEQ ID NO: 57), PIP-64Ea-2 (SEQ ID NO: 59), PIP-64Eb-2 (SEQID NO: 61), PIP-64Ec-2 (SEQ ID NO: 63), PIP-64Ha-2 (SEQ ID NO: 66),PIP-64Hb-2 (SEQ ID NO: 68), PIP-64Hc-2 (SEQ ID NO: 70), and PIP-64Hd-2(SEQ ID NO: 72). The amino acid diversity between the PIP-64-2polypeptide homologs is indicated with shading.

FIG. 5a-5b shows an alignment of the amino acid sequences of PIP-74Aa-1(SEQ ID NO: 73), PIP-74Ab-1 (SEQ ID NO: 75), and PIP-74Ca-1 (SEQ ID NO:77). The amino acid diversity between the PIP-74-1 polypeptide homologsis indicated with shading.

FIG. 6 shows an alignment of the amino acid sequences of PIP-74Aa-2 (SEQID NO: 74), PIP-74Ab-2 (SEQ ID NO: 76), and PIP-74Ca-2 (SEQ ID NO: 78).The amino acid diversity between PIP-74-2 polypeptide homologs isindicated with shading.

FIG. 7 shows an alignment of the amino acid sequences of PIP-75Aa (SEQID NO: 79), PIP-75Ba (SEQ ID NO: 80), PIP-75Da (SEQ ID NO: 81), PIP-75Ea(SEQ ID NO: 82), PIP-75Ga (SEQ ID NO: 83), PIP-75Gb (SEQ ID NO: 84),PIP-75Gc (SEQ ID NO: 85), PIP-75Gd (SEQ ID NO: 86), PIP-75Ge (SEQ ID NO:87). The amino acid diversity between the PIP-75 polypeptide homologs isindicated with shading.

FIG. 8a-8b shows an alignment of the amino acid sequences of PIP-77Aa(SEQ ID NO: 88, PIP-77Ab (SEQ ID NO: 89), PIP-77Ac (SEQ ID NO: 90),PIP-77Ad (SEQ ID NO: 91), PIP-77Ae (SEQ ID NO: 92), PIP-77Af (SEQ ID NO:240), PIP-77Ba (SEQ ID NO: 93), PIP-77Bb (SEQ ID NO: 94), PIP-77Bc (SEQID NO: 95), PIP-77Bd (SEQ ID NO: 96), PIP-77Be (SEQ ID NO: 97), PIP-77Bf(SEQ ID NO: 98), PIP-77Bg (SEQ ID NO: 99), PIP-77Bh (SEQ ID NO: 241),PIP-77Bi (SEQ ID NO: 242), PIP-77Ca (SEQ ID NO: 100), PIP-77Ea (SEQ IDNO: 101), PIP-77Eb (SEQ ID NO: 102), PIP-77Ec (SEQ ID NO: 103), PIP-77Ed(SEQ ID NO: 104), PIP-77Ee (SEQ ID NO: 105), PIP-77Ef (SEQ ID NO: 106),PIP-77Eg (SEQ ID NO: 107), PIP-77Eh (SEQ ID NO: 243), PIP-77Ei (SEQ IDNO: 244), and PIP-77Ej (SEQ ID NO: 245). The amino acid diversitybetween the PIP-77 polypeptide homologs is indicated with shading.

DETAILED DESCRIPTION

It is to be understood that this disclosure is not limited to theparticular methodology, protocols, cell lines, genera, and reagentsdescribed, as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentdisclosure.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisdisclosure belongs unless clearly indicated otherwise.

The present disclosure is drawn to compositions and methods forcontrolling pests. The methods involve transforming organisms withnucleic acid sequences encoding an insecticidal polypeptide of thedisclosure. In particular, the nucleic acid sequences of the embodimentsare useful for preparing plants and microorganisms that possesspesticidal activity. Thus, transformed bacteria, plants, plant cells,plant tissues and seeds are provided. The compositions are pesticidalnucleic acids and proteins of bacterial species. The nucleic acidsequences find use in the construction of expression vectors forsubsequent transformation into organisms of interest, as probes for theisolation of other homologous (or partially homologous) genes, and forthe generation of altered insecticidal polypeptides by methods known inthe art, such as site-directed mutagenesis, domain swapping or DNAshuffling. The insecticidal polypeptides of the disclosure find use incontrolling or killing Lepidopteran, Coleopteran, Dipteran, fungal,Hemipteran and nematode pest populations and for producing compositionswith pesticidal activity. Insect pests of interest include, but are notlimited to, Lepidoptera species including but not limited to:diamond-back moth, e.g., Helicoverpa zea Boddie; soybean looper, e.g.,Pseudoplusia includens Walker; and velvet bean caterpillar e.g.,Anticarsia gemmatalis Hübner and Coleoptera species including but notlimited to Western corn rootworm (Diabrotica virgifera)—WCRW, Southerncorn rootworm (Diabrotica undecimpunctata howardi)—SCRW, and Northerncorn rootworm (Diabrotica barberi)—NCRW.

By “pesticidal toxin” or “pesticidal protein” is used herein to refer toa toxin that has toxic activity against one or more pests, including,but not limited to, members of the Lepidoptera, Diptera, Hemiptera andColeoptera orders or the Nematoda phylum or a protein that has homologyto such a protein. Pesticidal proteins have been isolated from organismsincluding, for example, Bacillus sp., Pseudomonas sp., Photorhabdus sp.,Xenorhabdus sp., Clostridium bifermentans and Paenibacillus popilliae.Pesticidal proteins include but are not limited to: insecticidalproteins from Pseudomonas sp. such as PSEEN3174 (Monalysin; (2011) PLoSPathogens 7:1-13); from Pseudomonas protegens strain CHA0 and Pf-5(previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology10:2368-2386; GenBank Accession No. EU400157); from Pseudomonastaiwanensis (Liu, et al., (2010) J. Agric. Food Chem., 58:12343-12349)and from Pseudomonas pseudoalcligenes (Zhang, et al., (2009) Annals ofMicrobiology 59:45-50 and Li, et al., (2007) Plant Cell Tiss. OrganCult. 89:159-168); insecticidal proteins from Photorhabdus sp. andXenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxicology Journal,3:101-118 and Morgan, et al., (2001) Applied and Envir. Micro.67:2062-2069); U.S. Pat. Nos. 6,048,838, and 6,379,946; a PIP-1polypeptide of US Patent Publication Number US2014-0007292A1; an AflP-1Aand/or AflP-1B polypeptide(s) of US Patent Publication NumberUS2014-0033361; a PHI-4 polypeptides of U.S. Ser. No. 13/839,702; PIP-47polypeptides of of PCT Serial Number PCT/US14/51063; a PHI-4 polypeptideof US patent Publication US20140274885 or PCT Patent PublicationWO2014/150914; a PIP-72 polypeptide of PCT Serial Number PCT/US14/55128;the insecticidal proteins of U.S. Ser. No. 61/863,761 and 61/863,763;and δ-endotoxins including but not limited to: the Cry1, Cry2, Cry3,Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14,Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21, Cry22, Cry23, Cry24,Cry25, Cry26, Cry27, Cry28, Cry29, Cry30, Cry31, Cry32, Cry33, Cry34,Cry35, Cry36, Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44,Cry45, Cry 46, Cry47, Cry49, Cry 51, Cry52, Cry 53, Cry 54, Cry55,Cry56, Cry57, Cry58, Cry59. Cry60, Cry61, Cry62, Cry63, Cry64, Cry65,Cry66, Cry67, Cry68, Cry69, Cry70, Cry71 and Cry72 classes ofδ-endotoxin genes and the B. thuringiensis cytolytic cyt1 and cyt2genes. Members of these classes of B. thuringiensis insecticidalproteins include, but are not limited to Cry1Aa1 (Accession #AAA22353);Cry1Aa2 (Accession #Accession #AAA22552); Cry1Aa3 (Accession #BAA00257);Cry1Aa4 (Accession #CAA31886); Cry1Aa5 (Accession #BAA04468); Cry1Aa6(Accession #AAA86265); Cry1Aa7 (Accession #AAD46139); Cry1Aa8 (Accession#I26149); Cry1Aa9 (Accession #BAA77213); Cry1Aa10 (Accession #AAD55382);Cry1Aa11 (Accession #CAA70856); Cry1Aa12 (Accession #AAP80146); Cry1Aa13(Accession #AAM44305); Cry1Aa14 (Accession #AAP40639); Cry1Aa15(Accession #AAY66993); Cry1Aa16 (Accession #HQ439776); Cry1Aa17(Accession #HQ439788); Cry1Aa18 (Accession #HQ439790); Cry1Aa19(Accession #HQ685121); Cry1Aa20 (Accession #JF340156); Cry1Aa21(Accession #JN651496); Cry1Aa22 (Accession #KC158223); Cry1Ab1(Accession #AAA22330); Cry1Ab2 (Accession #AAA22613); Cry1Ab3 (Accession#AAA22561); Cry1Ab4 (Accession #BAA00071); Cry1Ab5 (Accession#CAA28405); Cry1Ab6 (Accession #AAA22420); Cry1Ab7 (Accession#CAA31620); Cry1Ab8 (Accession #AAA22551); Cry1Ab9 (Accession#CAA38701); Cry1Ab10 (Accession #A29125); Cry1Ab11 (Accession #I12419);Cry1Ab12 (Accession #AAC64003); Cry1Ab13 (Accession #AAN76494); Cry1Ab14(Accession #AAG16877); Cry1Ab15 (Accession #AAO13302); Cry1Ab16(Accession #AAK55546); Cry1Ab17 (Accession #AAT46415); Cry1Ab18(Accession #AAQ88259); Cry1Ab19 (Accession #AAW31761); Cry1Ab20(Accession #ABB72460); Cry1Ab21 (Accession #ABS18384); Cry1Ab22(Accession #ABW87320); Cry1Ab23 (Accession #HQ439777); Cry1Ab24(Accession #HQ439778); Cry1Ab25 (Accession #HQ685122); Cry1Ab26(Accession #HQ847729); Cry1Ab27 (Accession #JN135249); Cry1Ab28(Accession #JN135250); Cry1Ab29 (Accession #JN135251); Cry1Ab30(Accession #JN135252); Cry1Ab31 (Accession #JN135253); Cry1Ab32(Accession #JN135254); Cry1Ab33 (Accession #AAS93798); Cry1Ab34(Accession #KC156668); Cry1Ab-like (Accession #AAK14336); Cry1Ab-like(Accession #AAK14337); Cry1Ab-like (Accession #AAK14338); Cry1Ab-like(Accession #ABG88858); Cry1Ac1 (Accession #AAA22331); Cry1Ac2 (Accession#AAA22338); Cry1Ac3 (Accession #CAA38098); Cry1Ac4 (Accession#AAA73077); Cry1Ac5 (Accession #AAA22339); Cry1Ac6 (Accession#AAA86266); Cry1Ac7 (Accession #AAB46989); Cry1Ac8 (Accession#AAC44841); Cry1Ac9 (Accession #AAB49768); Cry1Ac10 (Accession#CAA05505); Cry1Ac11 (Accession #CAA10270); Cry1Ac12 (Accession#I12418); Cry1Ac13 (Accession #AAD38701); Cry1Ac14 (Accession#AAQ06607); Cry1Ac15 (Accession #AAN07788); Cry1Ac16 (Accession#AAU87037); Cry1Ac17 (Accession #AAX18704); Cry1Ac18 (Accession#AAY88347); Cry1Ac19 (Accession #ABD37053); Cry1Ac20 (Accession#ABB89046); Cry1Ac21 (Accession #AAY66992); Cry1Ac22 (Accession#ABZ01836); Cry1Ac23 (Accession #CA030431); Cry1Ac24 (Accession#ABL01535); Cry1Ac25 (Accession #FJ513324); Cry1Ac26 (Accession#FJ617446); Cry1Ac27 (Accession #FJ617447); Cry1Ac28 (Accession#ACM90319); Cry1Ac29 (Accession #D0438941); Cry1Ac30 (Accession#GQ227507); Cry1Ac31 (Accession #GU446674); Cry1Ac32 (Accession#HM061081); Cry1Ac33 (Accession #GQ866913); Cry1Ac34 (Accession#HQ230364); Cry1Ac35 (Accession #JF340157); Cry1Ac36 (Accession#JN387137); Cry1Ac37 (Accession #JQ317685); Cry1Ad1 (Accession#AAA22340); Cry1Ad2 (Accession #CAA01880); Cry1Ae1 (Accession#AAA22410); Cry1Af1 (Accession #AAB82749); Cry1Ag 1 (Accession#AAD46137); Cry1Ah1 (Accession #AAQ14326); Cry1Ah2 (Accession#ABB76664); Cry1Ah3 (Accession #HQ439779); Cry1Ai1 (Accession#AAO39719); Cry1Ai2 (Accession #HQ439780); Cry1A-like (Accession#AAK14339); Cry1Ba1 (Accession #CAA29898); Cry1Ba2 (Accession#CAA65003); Cry1Ba3 (Accession #AAK63251); Cry1Ba4 (Accession#AAK51084); Cry1Ba5 (Accession #AB020894); Cry1Ba6 (Accession#ABL60921); Cry1Ba7 (Accession #HQ439781); Cry1Bb1 (Accession#AAA22344); Cry1Bb2 (Accession #HQ439782); Cry1Bc1 (Accession#CAA86568); Cry1Bd1 (Accession #AAD10292); Cry1Bd2 (Accession#AAM93496); Cry1Be1 (Accession #AAC32850); Cry1Be2 (Accession#AAQ52387); Cry1Be3 (Accession #ACV96720); Cry1Be4 (Accession#HM070026); Cry1Bf1 (Accession #CAC50778); Cry1Bf2 (Accession#AAQ52380); Cry1Bg 1 (Accession #AAO39720); Cry1Bh1 (Accession#HQ589331); Cry1Bi1 (Accession #KC156700); Cry1Ca1 (Accession#CAA30396); Cry1Ca2 (Accession #CAA31951); Cry1Ca3 (Accession#AAA22343); Cry1Ca4 (Accession #CAA01886); Cry1Ca5 (Accession#CAA65457); Cry1Ca6 [1] (Accession #AAF37224); Cry1Ca1 (Accession#AAG50438); Cry1Ca8 (Accession #AAM00264); Cry1Ca9 (Accession#AAL79362); Cry1Ca10 (Accession #AAN16462); Cry1Ca11 (Accession#AAX53094); Cry1Ca12 (Accession #HM070027); Cry1Ca13 (Accession#HQ412621); Cry1Ca14 (Accession #JN651493); Cry1Cb1 (Accession #M97880);Cry1Cb2 (Accession #AAG35409); Cry1Cb3 (Accession #ACD50894);Cry1Cb-like (Accession #AAX63901); Cry1Da1 (Accession #CAA38099);Cry1Da2 (Accession #176415); Cry1Da3 (Accession #HQ439784); Cry1Db1(Accession #CAA80234); Cry1Db2 (Accession #AAK48937); Cry1Dc1 (Accession#ABK35074); Cry1Ea1 (Accession #CAA37933); Cry1Ea2 (Accession#CAA39609); Cry1Ea3 (Accession #AAA22345); Cry1Ea4 (Accession#AAD04732); Cry1Ea5 (Accession #A15535); Cry1Ea6 (Accession #AAL50330);Cry1Ea7 (Accession #AAW72936); Cry1Ea8 (Accession #ABX11258); Cry1Ea9(Accession #HQ439785); Cry1Ea10 (Accession #ADR00398); Cry1Ea11(Accession #JQ652456); Cry1Eb1 (Accession #AAA22346); Cry1Fa1 (Accession#AAA22348); Cry1Fa2 (Accession #AAA22347); Cry1Fa3 (Accession#HM070028); Cry1Fa4 (Accession #HM439638); Cry1Fb1 (Accession#CAA80235); Cry1Fb2 (Accession #BAA25298); Cry1Fb3 (Accession#AAF21767); Cry1Fb4 (Accession #AAC10641); Cry1Fb5 (Accession#AAO13295); Cry1Fb6 (Accession #ACD50892); Cry1Fb7 (Accession#ACD50893); Cry1Ga1 (Accession #CAA80233); Cry1Ga2 (Accession#CAA70506); Cry1Gb1 (Accession #AAD10291); Cry1Gb2 (Accession#AAO13756); Cry1Gc1 (Accession #AAQ52381); Cry1Ha1 (Accession#CAA80236); Cry1Hb1 (Accession #AAA79694); Cry1Hb2 (Accession#HQ439786); Cry1H-like (Accession #AAF01213); Cry1a1 (Accession#CAA44633); Cry1Ia2 (Accession #AAA22354); Cry1Ia3 (Accession#AAC36999); Cry1Ia4 (Accession #AAB00958); Cry1Ia5 (Accession#CAA70124); Cry1Ia6 (Accession #AAC26910); Cry1Ia7 (Accession#AAM73516); Cry1Ia8 (Accession #AAK66742); Cry1Ia9 (Accession#AAQ08616); Cry1Ia10 (Accession #AAP86782); Cry1Ia11 (Accession#CAC85964); Cry1Ia12 (Accession #AAV53390); Cry1Ia13 (Accession#ABF83202); Cry1Ia14 (Accession #ACG63871); Cry1Ia15 (Accession#FJ617445); Cry1Ia16 (Accession #FJ617448); Cry1Ia17 (Accession#GU989199); Cry1Ia18 (Accession #ADK23801); Cry1Ia19 (Accession#HQ439787); Cry1Ia20 (Accession #JQ228426); Cry1Ia21 (Accession#JQ228424); Cry1Ia22 (Accession #JQ228427); Cry1Ia23 (Accession#JQ228428); Cry1Ia24 (Accession #JQ228429); Cry1Ia25 (Accession#JQ228430); Cry1Ia26 (Accession #JQ228431); Cry1Ia27 (Accession#JQ228432); Cry1Ia28 (Accession #JQ228433); Cry1Ia29 (Accession#JQ228434); Cry1Ia30 (Accession #JQ317686); Cry1Ia31 (Accession#JX944038); Cry1Ia32 (Accession #JX944039); Cry1Ia33 (Accession#JX944040); Cry1Ib1 (Accession #AAA82114); Cry1Ib2 (Accession#ABW88019); Cry1Ib3 (Accession #ACD75515); Cry1Ib4 (Accession#HM051227); Cry1Ib5 (Accession #HM070028); Cry1Ib6 (Accession#ADK38579); Cry1Ib7 (Accession #JN571740); Cry1Ib8 (Accession#JN675714); Cry1Ib9 (Accession #JN675715); Cry1Ib10 (Accession#JN675716); Cry1Ib11 (Accession #JQ228423); Cry1Ic1 (Accession#AAC62933); Cry1Ic2 (Accession #AAE71691); Cry1Id1 (Accession#AAD44366); Cry1Id2 (Accession #JQ228422); Cry1Ie1 (Accession#AAG43526); Cry1Ie2 (Accession #HM439636); Cry1Ie3 (Accession#KC156647); Cry1Ie4 (Accession #KC156681); Cry1If1 (Accession#AAQ52382); Cry1Ig1 (Accession #KC156701); Cry1I-like (Accession#AAC31094); Cry1I-like (Accession #ABG88859); Cry1Ja1 (Accession#AAA22341); Cry1Ja2 (Accession #HM070030); Cry1Ja3 (Accession#JQ228425); Cry1Jb1 (Accession #AAA98959); Cry1Jc1 (Accession#AAC31092); Cry1Jc2 (Accession #AAQ52372); Cry1Jd1 (Accession#CAC50779); Cry1Ka1 (Accession #AAB00376); Cry1Ka2 (Accession#HQ439783); Cry1La1 (Accession #AAS60191); Cry1La2 (Accession#HM070031); Cry1Ma1 (Accession #FJ884067); Cry1Ma2 (Accession#KC156659); Cry1Na1 (Accession #KC156648); Cry1Nb1 (Accession#KC156678); Cry1-like (Accession #AAC31091); Cry2Aa1 (Accession#AAA22335); Cry2Aa2 (Accession #AAA83516); Cry2Aa3 (Accession #D86064);Cry2Aa4 (Accession #AAC04867); Cry2Aa5 (Accession #CAA10671); Cry2Aa6(Accession #CAA10672); Cry2Aa7 (Accession #CAA10670); Cry2Aa8 (Accession#AAO13734); Cry2Aa9 (Accession #AAO13750); Cry2Aa10 (Accession#AAQ04263); Cry2Aa11 (Accession #AAQ52384); Cry2Aa12 (Accession#AB183671); Cry2Aa13 (Accession #ABL01536); Cry2Aa14 (Accession#ACF04939); Cry2Aa15 (Accession #JN426947); Cry2Ab1 (Accession#AAA22342); Cry2Ab2 (Accession #CAA39075); Cry2Ab3 (Accession#AAG36762); Cry2Ab4 (Accession #AAO13296); Cry2Ab5 (Accession#AAQ04609); Cry2Ab6 (Accession #AAP59457); Cry2Ab7 (Accession#AAZ66347); Cry2Ab8 (Accession #ABC95996); Cry2Ab9 (Accession#ABC74968); Cry2Ab10 (Accession #EF157306); Cry2Ab11 (Accession#CAM84575); Cry2Ab12 (Accession #ABM21764); Cry2Ab13 (Accession#ACG76120); Cry2Ab14 (Accession #ACG76121); Cry2Ab15 (Accession#HM037126); Cry2Ab16 (Accession #GQ866914); Cry2Ab17 (Accession#HQ439789); Cry2Ab18 (Accession #JN135255); Cry2Ab19 (Accession#JN135256); Cry2Ab20 (Accession #JN135257); Cry2Ab21 (Accession#JN135258); Cry2Ab22 (Accession #JN135259); Cry2Ab23 (Accession#JN135260); Cry2Ab24 (Accession #JN135261); Cry2Ab25 (Accession#JN415485); Cry2Ab26 (Accession #JN426946); Cry2Ab27 (Accession#JN415764); Cry2Ab28 (Accession #JN651494); Cry2Ac1 (Accession#CAA40536); Cry2Ac2 (Accession #AAG35410); Cry2Ac3 (Accession#AAQ52385); Cry2Ac4 (Accession #ABC95997); Cry2Ac5 (Accession#ABC74969); Cry2Ac6 (Accession #ABC74793); Cry2Ac7 (Accession#CAL18690); Cry2Ac8 (Accession #CAM09325); Cry2Ac9 (Accession#CAM09326); Cry2Ac10 (Accession #ABN15104); Cry2Ac11 (Accession#CAM83895); Cry2Ac12 (Accession #CAM83896); Cry2Ad1 (Accession#AAF09583); Cry2Ad2 (Accession #ABC86927); Cry2Ad3 (Accession#CAK29504); Cry2Ad4 (Accession #CAM32331); Cry2Ad5 (Accession#CA078739); Cry2Ae1 (Accession #AAQ52362); Cry2Af1 (Accession#AB030519); Cry2Af2 (Accession #GQ866915); Cry2Ag1 (Accession#ACH91610); Cry2Ah1 (Accession #EU939453); Cry2Ah2 (Accession#ACL80665); Cry2Ah3 (Accession #GU073380); Cry2Ah4 (Accession#KC156702); Cry2Ai1 (Accession #FJ788388); Cry2Aj (Accession #); Cry2Ak1(Accession #KC156660); Cry2Ba1 (Accession #KC156658); Cry3Aa1 (Accession#AAA22336); Cry3Aa2 (Accession #AAA22541); Cry3Aa3 (Accession#CAA68482); Cry3Aa4 (Accession #AAA22542); Cry3Aa5 (Accession#AAA50255); Cry3Aa6 (Accession #AAC43266); Cry3Aa7 (Accession#CAB41411); Cry3Aa8 (Accession #AAS79487); Cry3Aa9 (Accession#AAW05659); Cry3Aa10 (Accession #AAU29411); Cry3Aa11 (Accession#AAW82872); Cry3Aa12 (Accession #ABY49136); Cry3Ba1 (Accession#CAA34983); Cry3Ba2 (Accession #CAA00645); Cry3Ba3 (Accession#JQ397327); Cry3Bb1 (Accession #AAA22334); Cry3Bb2 (Accession#AAA74198); Cry3Bb3 (Accession #I15475); Cry3Ca1 (Accession #CAA42469);Cry4Aa1 (Accession #CAA68485); Cry4Aa2 (Accession #BAA00179); Cry4Aa3(Accession #CAD30148); Cry4Aa4 (Accession #AFB18317); Cry4A-like(Accession #AAY96321); Cry4Ba1 (Accession #CAA30312); Cry4Ba2 (Accession#CAA30114); Cry4Ba3 (Accession #AAA22337); Cry4Ba4 (Accession#BAA00178); Cry4Ba5 (Accession #CAD30095); Cry4Ba-like (Accession#ABC47686); Cry4Ca1 (Accession #EU646202); Cry4Cb1 (Accession#FJ403208); Cry4Cb2 (Accession #FJ597622); Cry4Cc1 (Accession#FJ403207); Cry5Aa1 (Accession #AAA67694); Cry5Ab1 (Accession#AAA67693); Cry5Ac1 (Accession #I34543); Cry5Ad1 (Accession #ABQ82087);Cry5Ba1 (Accession #AAA68598); Cry5Ba2 (Accession #ABW88931); Cry5Ba3(Accession #AFJ04417); Cry5Ca1 (Accession #HM461869); Cry5Ca2 (Accession#ZP_04123426); Cry5Da1 (Accession #HM461870); Cry5Da2 (Accession#ZP_04123980); Cry5Ea1 (Accession #HM485580); Cry5Ea2 (Accession#ZP_04124038); Cry6Aa1 (Accession #AAA22357); Cry6Aa2 (Accession#AAM46849); Cry6Aa3 (Accession #ABH03377); Cry6Ba1 (Accession#AAA22358); Cry7Aa1 (Accession #AAA22351); Cry7Ab1 (Accession#AAA21120); Cry7Ab2 (Accession #AAA21121); Cry7Ab3 (Accession#ABX24522); Cry7Ab4 (Accession #EU380678); Cry7Ab5 (Accession#ABX79555); Cry7Ab6 (Accession #AC144005); Cry7Ab7 (Accession#ADB89216); Cry7Ab8 (Accession #GU145299); Cry7Ab9 (Accession#ADD92572); Cry7Ba1 (Accession #ABB70817); Cry7Bb1 (Accession#KC156653); Cry7Ca1 (Accession #ABR67863); Cry7Cb1 (Accession#KC156698); Cry7Da1 (Accession #ACQ99547); Cry7Da2 (Accession#HM572236); Cry7Da3 (Accession #KC156679); Cry7Ea1 (Accession#HM035086); Cry7Ea2 (Accession #HM132124); Cry7Ea3 (Accession#EEM19403); Cry7Fa1 (Accession #HM035088); Cry7Fa2 (Accession#EEM19090); Cry7Fb1 (Accession #HM572235); Cry7Fb2 (Accession#KC156682); Cry7Ga1 (Accession #HM572237); Cry7Ga2 (Accession#KC156669); Cry7Gb1 (Accession #KC156650); Cry7Gc1 (Accession#KC156654); Cry7Gd1 (Accession #KC156697); Cry7Ha1 (Accession#KC156651); Cry71a1 (Accession #KC156665); Cry7Ja1 (Accession#KC156671); Cry7Ka1 (Accession #KC156680); Cry7Kb1 (Accession#BAM99306); Cry7La1 (Accession #BAM99307); Cry8Aa1 (Accession#AAA21117); Cry8Ab1 (Accession #EU044830); Cry8Ac1 (Accession#KC156662); Cry8Ad1 (Accession #KC156684); Cry8Ba1 (Accession#AAA21118); Cry8Bb1 (Accession #CAD57542); Cry8Bc1 (Accession#CAD57543); Cry8Ca1 (Accession #AAA21119); Cry8Ca2 (Accession#AAR98783); Cry8Ca3 (Accession #EU625349); Cry8Ca4 (Accession#ADB54826); Cry8Da1 (Accession #BAC07226); Cry8Da2 (Accession#BD133574); Cry8Da3 (Accession #BD133575); Cry8Db1 (Accession#BAF93483); Cry8Ea1 (Accession #AAQ73470); Cry8Ea2 (Accession#EU047597); Cry8Ea3 (Accession #KC855216); Cry8Fa1 (Accession#AAT48690); Cry8Fa2 (Accession #HQ174208); Cry8Fa3 (Accession#AFH78109); Cry8Ga1 (Accession #AAT46073); Cry8Ga2 (Accession#ABC42043); Cry8Ga3 (Accession #FJ198072); Cry8Ha1 (Accession#AAW81032); Cry8Ia1 (Accession #EU381044); Cry8Ia2 (Accession#GU073381); Cry8Ia3 (Accession #HM044664); Cry8Ia4 (Accession#KC156674); Cry8Ib1 (Accession #GU325772); Cry8Ib2 (Accession#KC156677); Cry8Ja1 (Accession #EU625348); Cry8Ka1 (Accession#FJ422558); Cry8Ka2 (Accession #ACN87262); Cry8Kb1 (Accession#HM123758); Cry8Kb2 (Accession #KC156675); Cry8La1 (Accession#GU325771); Cry8Ma1 (Accession #HM044665); Cry8Ma2 (Accession#EEM86551); Cry8Ma3 (Accession #HM210574); Cry8Na1 (Accession#HM640939); Cry8Pa1 (Accession #HQ388415); Cry8Qa1 (Accession#HQ441166); Cry8Qa2 (Accession #KC152468); Cry8Ra1 (Accession#AFP87548); Cry8Sa1 (Accession #JQ740599); Cry8Ta1 (Accession#KC156673); Cry8-like (Accession #FJ770571); Cry8-like (Accession#ABS53003); Cry9Aa1 (Accession #CAA41122); Cry9Aa2 (Accession#CAA41425); Cry9Aa3 (Accession #GQ249293); Cry9Aa4 (Accession#GQ249294); Cry9Aa5 (Accession #JX174110); Cry9Aa like (Accession#AAQ52376); Cry9Ba1 (Accession #CAA52927); Cry9Ba2 (Accession#GU299522); Cry9Bb1 (Accession #AAV28716); Cry9Ca1 (Accession#CAA85764); Cry9Ca2 (Accession #AAQ52375); Cry9Da1 (Accession#BAA19948); Cry9Da2 (Accession #AAB97923); Cry9Da3 (Accession#GQ249293); Cry9Da4 (Accession #GQ249297); Cry9Db1 (Accession#AAX78439); Cry9Dc1 (Accession #KC156683); Cry9Ea1 (Accession#BAA34908); Cry9Ea2 (Accession #AAO12908); Cry9Ea3 (Accession#ABM21765); Cry9Ea4 (Accession #ACE88267); Cry9Ea5 (Accession#ACF04743); Cry9Ea6 (Accession #ACG63872); Cry9Ea7 (Accession#FJ380927); Cry9Ea8 (Accession #GQ249292); Cry9Ea9 (Accession#JN651495); Cry9Eb1 (Accession #CAC50780); Cry9Eb2 (Accession#GQ249298); Cry9Eb3 (Accession #KC156646); Cry9Ec1 (Accession#AAC63366); Cry9Ed1 (Accession #AAX78440); Cry9Ee1 (Accession#GQ249296); Cry9Ee2 (Accession #KC156664); Cry9Fa1 (Accession#KC156692); Cry9Ga1 (Accession #KC156699); Cry9-like (Accession#AAC63366); Cry10Aa1 (Accession #AAA22614); Cry10Aa2 (Accession#E00614); Cry10Aa3 (Accession #CAD30098); Cry10Aa4 (Accession#AFB18318); Cry10A-like (Accession #D0167578); Cry11Aa1 (Accession#AAA22352); Cry11Aa2 (Accession #AAA22611); Cry11Aa3 (Accession#CAD30081); Cry11Aa4 (Accession #AFB18319); Cry11Aa-like (Accession#DQ166531); Cry11Ba1 (Accession #CAA60504); Cry11 Bb1 (Accession#AAC97162); Cry11Bb2 (Accession #HM068615); Cry12Aa1 (Accession#AAA22355); Cry13Aa1 (Accession #AAA22356); Cry14Aa1 (Accession#AAA21516); Cry14Ab1 (Accession #KC156652); Cry15Aa1 (Accession#AAA22333); Cry16Aa1 (Accession #CAA63860); Cry17Aa1 (Accession#CAA67841); Cry18Aa1 (Accession #CAA67506); Cry18Ba1 (Accession#AAF89667); Cry18Ca1 (Accession #AAF89668); Cry19Aa1 (Accession#CAA68875); Cry19Ba1 (Accession #BAA32397); Cry19Ca1 (Accession#AFM37572); Cry20Aa1 (Accession #AAB93476); Cry20Ba1 (Accession#ACS93601); Cry20Ba2 (Accession #KC156694); Cry20-like (Accession#GQ144333); Cry21Aa1 (Accession #I32932); Cry21Aa2 (Accession #I66477);Cry21Ba1 (Accession #BAC06484); Cry21Ca1 (Accession #JF521577); Cry21Ca2(Accession #KC156687); Cry21Da1 (Accession #JF521578); Cry22Aa1(Accession #I34547); Cry22Aa2 (Accession #CAD43579); Cry22Aa3 (Accession#ACD93211); Cry22Ab1 (Accession #AAK50456); Cry22Ab2 (Accession#CAD43577); Cry22Ba1 (Accession #CAD43578); Cry22Bb1 (Accession#KC156672); Cry23Aa1 (Accession #AAF76375); Cry24Aa1 (Accession#AAC61891); Cry24Ba1 (Accession #BAD32657); Cry24Ca1 (Accession#CAJ43600); Cry25Aa1 (Accession #AAC61892); Cry26Aa1 (Accession#AAD25075); Cry27Aa1 (Accession #BAA82796); Cry28Aa1 (Accession#AAD24189); Cry28Aa2 (Accession #AAG00235); Cry29Aa1 (Accession#CAC80985); Cry30Aa1 (Accession #CAC80986); Cry30Ba1 (Accession#BAD00052); Cry30Ca1 (Accession #BAD67157); Cry30Ca2 (Accession#ACU24781); Cry30Da1 (Accession #EF095955); Cry30Db1 (Accession#BAE80088); Cry30Ea1 (Accession #ACC95445); Cry30Ea2 (Accession#FJ499389); Cry30Fa1 (Accession #ACI22625); Cry30Ga1 (Accession#ACG60020); Cry30Ga2 (Accession #HQ638217); Cry31Aa1 (Accession#BAB11757); Cry31Aa2 (Accession #AAL87458); Cry31Aa3 (Accession#BAE79808); Cry31Aa4 (Accession #BAF32571); Cry31Aa5 (Accession#BAF32572); Cry31Aa6 (Accession #BA144026); Cry31Ab1 (Accession#BAE79809); Cry31Ab2 (Accession #BAF32570); Cry31Ac1 (Accession#BAF34368); Cry31Ac2 (Accession #AB731600); Cry31Ad1 (Accession#BA144022); Cry32Aa1 (Accession #AAG36711); Cry32Aa2 (Accession#GU063849); Cry32Ab1 (Accession #GU063850); Cry32Ba1 (Accession#BAB78601); Cry32Ca1 (Accession #BAB78602); Cry32Cb1 (Accession#KC156708); Cry32Da1 (Accession #BAB78603); Cry32Ea1 (Accession#GU324274); Cry32Ea2 (Accession #KC156686); Cry32Eb1 (Accession#KC156663); Cry32Fa1 (Accession #KC156656); Cry32Ga1 (Accession#KC156657); Cry32Ha1 (Accession #KC156661); Cry32Hb1 (Accession#KC156666); Cry32Ia1 (Accession #KC156667); Cry32Ja1 (Accession#KC156685); Cry32Ka1 (Accession #KC156688); Cry32La1 (Accession#KC156689); Cry32Ma1 (Accession #KC156690); Cry32Mb1 (Accession#KC156704); Cry32Na1 (Accession #KC156691); Cry32Oa1 (Accession#KC156703); Cry32Pa1 (Accession #KC156705); Cry32Qa1 (Accession#KC156706); Cry32Ra1 (Accession #KC156707); Cry32Sa1 (Accession#KC156709); Cry32Ta1 (Accession #KC156710); Cry32Ua1 (Accession#KC156655); Cry33Aa1 (Accession #AAL26871); Cry34Aa1 (Accession#AAG50341); Cry34Aa2 (Accession #AAK64560); Cry34Aa3 (Accession#AAT29032); Cry34Aa4 (Accession #AAT29030); Cry34Ab1 (Accession#AAG41671); Cry34Ac1 (Accession #AAG50118); Cry34Ac2 (Accession#AAK64562); Cry34Ac3 (Accession #AAT29029); Cry34Ba1 (Accession#AAK64565); Cry34Ba2 (Accession #AAT29033); Cry34Ba3 (Accession#AAT29031); Cry35Aa1 (Accession #AAG50342); Cry35Aa2 (Accession#AAK64561); Cry35Aa3 (Accession #AAT29028); Cry35Aa4 (Accession#AAT29025); Cry35Ab1 (Accession #AAG41672); Cry35Ab2 (Accession#AAK64563); Cry35Ab3 (Accession #AY536891); Cry35Ac1 (Accession#AAG50117); Cry35Ba1 (Accession #AAK64566); Cry35Ba2 (Accession#AAT29027); Cry35Ba3 (Accession #AAT29026); Cry36Aa1 (Accession#AAK64558); Cry37Aa1 (Accession #AAF76376); Cry38Aa1 (Accession#AAK64559); Cry39Aa1 (Accession #BAB72016); Cry40Aa1 (Accession#BAB72018); Cry40Ba1 (Accession #BAC77648); Cry40Ca1 (Accession#EU381045); Cry40Da1 (Accession #ACF15199); Cry41Aa1 (Accession#BAD35157); Cry41Ab1 (Accession #BAD35163); Cry41Ba1 (Accession#HM461871); Cry41Ba2 (Accession #ZP_04099652); Cry42Aa1 (Accession#BAD35166); Cry43Aa1 (Accession #BAD15301); Cry43Aa2 (Accession#BAD95474); Cry43Ba1 (Accession #BAD15303); Cry43Ca1 (Accession#KC156676); Cry43Cb1 (Accession #KC156695); Cry43Cc1 (Accession#KC156696); Cry43-like (Accession #BAD15305); Cry44Aa (Accession#BAD08532); Cry45Aa (Accession #BAD22577); Cry46Aa (Accession#BAC79010); Cry46Aa2 (Accession #BAG68906); Cry46Ab (Accession#BAD35170); Cry47Aa (Accession #AAY24695); Cry48Aa (Accession#CAJ18351); Cry48Aa2 (Accession #CAJ86545); Cry48Aa3 (Accession#CAJ86546); Cry48Ab (Accession #CAJ86548); Cry48Ab2 (Accession#CAJ86549); Cry49Aa (Accession #CAH56541); Cry49Aa2 (Accession#CAJ86541); Cry49Aa3 (Accession #CAJ86543); Cry49Aa4 (Accession#CAJ86544); Cry49Ab1 (Accession #CAJ86542); Cry50Aa1 (Accession#BAE86999); Cry50Ba1 (Accession #GU446675); Cry50Ba2 (Accession#GU446676); Cry51Aa1 (Accession #AB114444); Cry51Aa2 (Accession#GU570697); Cry52Aa1 (Accession #EF613489); Cry52Ba1 (Accession#FJ361760); Cry53Aa1 (Accession #EF633476); Cry53Ab1 (Accession#FJ361759); Cry54Aa1 (Accession #ACA52194); Cry54Aa2 (Accession#GQ140349); Cry54Ba1 (Accession #GU446677); Cry55Aa1 (Accession#ABW88932); Cry54Ab1 (Accession #JQ916908); Cry55Aa2 (Accession#AAE33526); Cry56Aa1 (Accession #ACU57499); Cry56Aa2 (Accession#GQ483512); Cry56Aa3 (Accession #JX025567); Cry57Aa1 (Accession#ANC87261); Cry58Aa1 (Accession #ANC87260); Cry59Ba1 (Accession#JN790647); Cry59Aa1 (Accession #ACR43758); Cry60Aa1 (Accession#ACU24782); Cry60Aa2 (Accession #EAO57254); Cry60Aa3 (Accession#EEM99278); Cry60Ba1 (Accession #GU810818); Cry60Ba2 (Accession#EAO57253); Cry60Ba3 (Accession #EEM99279); Cry61Aa1 (Accession#HM035087); Cry61Aa2 (Accession #HM132125); Cry61Aa3 (Accession#EEM19308); Cry62Aa1 (Accession #HM054509); Cry63Aa1 (Accession#BA144028); Cry64Aa1 (Accession #BAJ05397); Cry65Aa1 (Accession#HM461868); Cry65Aa2 (Accession #ZP_04123838); Cry66Aa1 (Accession#HM485581); Cry66Aa2 (Accession #ZP_04099945); Cry67Aa1 (Accession#HM485582); Cry67Aa2 (Accession #ZP_04148882); Cry68Aa1 (Accession#HQ113114); Cry69Aa1 (Accession #HQ401006); Cry69Aa2 (Accession#JQ821388); Cry69Ab1 (Accession #JN209957); Cry70Aa1 (Accession#JN646781); Cry70Ba1 (Accession #AD051070); Cry70Bb1 (Accession#EEL67276); Cry71Aa1 (Accession #JX025568); Cry72Aa1 (Accession#JX025569); Cyt1Aa (GenBank Accession Number X03182); Cyt1Ab (GenBankAccession Number X98793); Cyt1B (GenBank Accession Number U37196); Cyt2A(GenBank Accession Number Z14147); and Cyt2B (GenBank Accession NumberU52043).

Examples of δ-endotoxins also include but are not limited to Cry1Aproteins of U.S. Pat. Nos. 5,880,275 and 7,858,849; a DIG-3 or DIG-11toxin (N-terminal deletion of α-helix 1 and/or α-helix 2 variants of cryproteins such as Cry1A, Cry3A) of U.S. Pat. Nos. 8,304,604, 8.304,605and 8,476,226; Cry1B of U.S. patent application Ser. No. 10/525,318;Cry1C of U.S. Pat. No. 6,033,874; Cry1F of U.S. Pat. Nos. 5,188,960 and6,218,188; Cry1A/F chimeras of U.S. Pat. Nos. 7,070,982; 6,962,705 and6,713,063); a Cry2 protein such as Cry2Ab protein of U.S. Pat. No.7,064,249); a Cry3A protein including but not limited to an engineeredhybrid insecticidal protein (eHIP) created by fusing unique combinationsof variable regions and conserved blocks of at least two different Cryproteins (US Patent Application Publication Number 2010/0017914); a Cry4protein; a Cry5 protein; a Cry6 protein; Cry8 proteins of U.S. Pat. Nos.7,329,736, 7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,378,499 and7,462,760; a Cry9 protein such as such as members of the Cry9A, Cry9B,Cry9C, Cry9D, Cry9E and Cry9F families; a Cry15 protein of Naimov, etal., (2008) Applied and Environmental Microbiology, 74:7145-7151; aCry22, a Cry34Ab1 protein of U.S. Pat. Nos. 6,127,180, 6,624,145 and6,340,593; a CryET33 and cryET34 protein of U.S. Pat. Nos. 6,248,535,6,326,351, 6,399,330, 6,949,626, 7,385,107 and 7,504,229; a CryET33 andCryET34 homologs of US Patent Publication Number 2006/0191034,2012/0278954, and PCT Publication Number WO 2012/139004; a Cry35Ab1protein of U.S. Pat. Nos. 6,083,499, 6,548,291 and 6,340,593; a Cry46protein, a Cry 51 protein, a Cry binary toxin; a TIC901 or relatedtoxin; TIC807 of US Patent Application Publication Number 2008/0295207;ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128 of PCT US2006/033867; TIC853 toxins of U.S. Pat. No. 8,513,494, AXMI-027,AXMI-036, and AXMI-038 of U.S. Pat. No. 8,236,757; AXMI-031, AXMI-039,AXMI-040, AXMI-049 of U.S. Pat. No. 7,923,602; AXMI-018, AXMI-020 andAXMI-021 of WO 2006/083891; AXMI-010 of WO 2005/038032; AXMI-003 of WO2005/021585; AXMI-008 of US Patent Application Publication Number2004/0250311; AXMI-006 of US Patent Application Publication Number2004/0216186; AXMI-007 of US Patent Application Publication Number2004/0210965; AXMI-009 of US Patent Application Number 2004/0210964;AXMI-014 of US Patent Application Publication Number 2004/0197917;AXMI-004 of US Patent Application Publication Number 2004/0197916;AXMI-028 and AXMI-029 of WO 2006/119457; AXMI-007, AXMI-008,AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO 2004/074462;AXMI-150 of U.S. Pat. No. 8,084,416; AXMI-205 of US Patent ApplicationPublication Number 2011/0023184; AXMI-011, AXMI-012, AXMI-013, AXMI-015,AXMI-019, AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022,AXMI-023, AXMI-041, AXMI-063 and AXMI-064 of US Patent ApplicationPublication Number 2011/0263488; AXMI-R1 and related proteins of USPatent Application Publication Number 2010/0197592; AXMI221Z, AXMI222z,AXMI223z, AXMI224z and AXMI225z of WO 2011/103248; AXMI218, AXMI219,AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230 and AXMI231 of WO2011/103247; AXMI-115, AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of U.S.Pat. No. 8,334,431; AXMI-001, AXMI-002, AXMI-030, AXMI-035 and AXMI-045of US Patent Application Publication Number 2010/0298211; AXMI-066 andAXMI-076 of US Patent Application Publication Number 2009/0144852;AXMI128, AXMI130, AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143,AXMI144, AXMI146, AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155,AXMI156, AXMI157, AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI168,AXMI169, AXMI170, AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, AXMI176,AXMI177, AXMI178, AXMI179, AXMI180, AXMI181, AXMI182, AXMI185, AXMI186,AXMI187, AXMI188, AXMI189 of U.S. Pat. No. 8,318,900; AXMI079, AXMI080,AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099,AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109,AXMI110, AXMI111, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119,AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268,AXMI127, AXMI129, AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138,AXMI137 of US Patent Application Publication Number 2010/0005543, cryproteins such as Cry1A and Cry3A having modified proteolytic sites ofU.S. Pat. No. 8,319,019; a Cry1Ac, Cry2Aa and Cry1Ca toxin protein fromBacillus thuringiensis strain VBTS 2528 of US Patent ApplicationPublication Number 2011/0064710. Other Cry proteins are well known toone skilled in the art (see, Crickmore, et al., “Bacillus thuringiensistoxin nomenclature” (2011), at lifesci.sussex.ac.uk/home/NeilCrickmore/Bt/ which can be accessed on the world-wide web using the“www” prefix). The insecticidal activity of Cry proteins is well knownto one skilled in the art (for review, see, van Frannkenhuyzen, (2009)J. Invert. Path. 101:1-16). The use of Cry proteins as transgenic planttraits is well known to one skilled in the art and Cry-transgenic plantsincluding but not limited to plants expressing Cry1Ac, Cry1Ac+Cry2Ab,Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A,Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c and CBI-Bt havereceived regulatory approval (see, Sanahuja, (2011) Plant BiotechJournal 9:283-300 and the CERA (2010) GM Crop Database Center forEnvironmental Risk Assessment (CERA), ILSI Research Foundation,Washington, D.C. at cera-gmc.org/index.php?action=gm_crop_database,which can be accessed on the world-wide web using the “www” prefix).More than one pesticidal proteins well known to one skilled in the artcan also be expressed in plants such as Vip3Ab & Cry1Fa(US2012/0317682); Cry1BE & Cry1F (US2012/0311746); Cry1CA & Cry1AB(US2012/0311745); Cry1F & CryCa (US2012/0317681); Cry1DA & Cry1BE(US2012/0331590); Cry1DA & Cry1Fa (US2012/0331589); Cry1AB & Cry1BE(US2012/0324606); Cry1Fa & Cry2Aa and Cry1I & Cry1E (US2012/0324605);Cry34Ab/35Ab and Cry6Aa (US20130167269); Cry34Ab/VCry35Ab & Cry3Aa(US20130167268); and Cry3A and Cry1Ab or Vip3Aa (US20130116170).Pesticidal proteins also include insecticidal lipases including lipidacyl hydrolases of U.S. Pat. No. 7,491,869, and cholesterol oxidasessuch as from Streptomyces (Purcell et al. (1993) Biochem Biophys ResCommun 15:1406-1413). Pesticidal proteins also include VIP (vegetativeinsecticidal proteins) toxins of U.S. Pat. Nos. 5,877,012, 6,107,2796,137,033, 7,244,820, 7,615,686, and 8,237,020 and the like. Other VIPproteins are well known to one skilled in the art (see,Iifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html which can beaccessed on the world-wide web using the “www” prefix). Pesticidalproteins also include toxin complex (TC) proteins, obtainable fromorganisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, U.S.Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have “stand alone”insecticidal activity and other TC proteins enhance the activity of thestand-alone toxins produced by the same given organism. The toxicity ofa “stand-alone” TC protein (from Photorhabdus, Xenorhabdus orPaenibacillus, for example) can be enhanced by one or more TC protein“potentiators” derived from a source organism of a different genus.There are three main types of TC proteins. As referred to herein, ClassA proteins (“Protein A”) are stand-alone toxins. Class B proteins(“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity ofClass A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 andXptA2. Examples of Class B proteins are TcaC, TcdB, XptB1Xb and XptC1Wi.Examples of Class C proteins are TccC, XptC1Xb and XptB1Wi. Pesticidalproteins also include spider, snake and scorpion venom proteins.Examples of spider venom peptides include but not limited to lycotoxin-1peptides and mutants thereof (U.S. Pat. No. 8,334,366).

In some embodiments the insecticidal polypeptides of the disclosureinclude amino acid sequences deduced from the full-length nucleic acidsequences disclosed herein and amino acid sequences that are shorterthan the full-length sequences, either due to the use of an alternatedownstream start site or due to processing that produces a shorterprotein having pesticidal activity. Processing may occur in the organismthe protein is expressed in or in the pest after ingestion of theprotein.

Thus, provided herein are novel isolated or recombinant nucleic acidsequences that confer pesticidal activity. Also provided are the aminoacid sequences of insecticidal polypeptides of the disclosure. Theprotein resulting from translation of these insecticidal polypeptidegenes allows cells to control or kill pests that ingest it.

Nucleic Acid Molecules, and Variants and Fragments Thereof

One aspect of the disclosure pertains to isolated or recombinant nucleicacid molecules comprising nucleic acid sequences encoding insecticidalpolypeptides of the disclosure or biologically active portions thereof,as well as nucleic acid molecules sufficient for use as hybridizationprobes to identify nucleic acid molecules encoding proteins with regionsof sequence homology. As used herein, the term “nucleic acid molecule”refers to DNA molecules (e.g., recombinant DNA, cDNA, genomic DNA,plastid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

An “isolated” nucleic acid molecule (or DNA) is used herein to refer toa nucleic acid sequence (or DNA) that is no longer in its naturalenvironment, for example in vitro. A “recombinant” nucleic acid molecule(or DNA) is used herein to refer to a nucleic acid sequence (or DNA)that is in a recombinant bacterial or plant host cell. In someembodiments, an “isolated” or “recombinant” nucleic acid is free ofsequences (preferably protein encoding sequences) that naturally flankthe nucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For purposes of the disclosure, “isolated” or“recombinant” when used to refer to nucleic acid molecules excludesisolated chromosomes. For example, in various embodiments, therecombinant nucleic acid molecule encoding an insecticidal polypeptidecan contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1kb of nucleic acid sequences that naturally flank the nucleic acidmolecule in genomic DNA of the cell from which the nucleic acid isderived.

In some embodiments an isolated nucleic acid molecule encoding aninsecticidal polypeptide of the disclosure has one or more change in thenucleic acid sequence compared to the native or genomic nucleic acidsequence. In some embodiments the change in the native or genomicnucleic acid sequence includes but is not limited to: changes in thenucleic acid sequence due to the degeneracy of the genetic code; changesin the nucleic acid sequence due to the amino acid substitution,insertion, deletion and/or addition compared to the native or genomicsequence; removal of one or more intron; deletion of one or moreupstream or downstream regulatory regions; and deletion of the 5′ and/or3′ untranslated region associated with the genomic nucleic acidsequence. In some embodiments the nucleic acid molecule encoding aninsecticidal polypeptide is a non-genomic sequence.

Polynucleotides encoding PIP-45-1 polypeptides are encompassed by thedisclosure. A variety of polynucleotides encoding PIP-45-1 polypeptidesare contemplated. One source of a polynucleotide encoding a PIP-45-1polypeptide or related proteins is a bacterial strain that contains thepolynucleotide of SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 126, SEQ IDNO: 128, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138,SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ IDNO: 152, SEQ ID NO: 220 or SEQ ID NO: 222 that encode the PIP-45-1polypeptide of SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ IDNO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45, SEQID NO: 234 and SEQ ID NO: 236, respectively. One source of apolynucleotide encoding a PIP-45-1 polypeptide or related proteins isfrom a Pseudomonas, Thalassuspira, Paracoccus or Cellvibrio strain. Onesource of a polynucleotide encoding a PIP-45-1 polypeptide or relatedproteins is from a Pseudomonas strain selected from but not limited toPseudomonas brenneri, Pseudomonas monteilii, Pseudomonas gessardii,Pseudomonas plecoglossicida, Pseudomonas putida, Pseudomonas poae,Pseudomonas trivialis, Pseudomonas libanensis, Pseudomonas fluorescensand Pseudomonas asplenii.

In some embodiments the nucleic acid molecule encoding the PIP-45-1polypeptide is a non-genomic nucleic acid sequence. As used herein a“non-genomic nucleic acid sequence” or “non-genomic nucleic acidmolecule” or “non-genomic polynucleotide” refers to a nucleic acidmolecule that has one or more change in the nucleic acid sequencecompared to a native or genomic nucleic acid sequence. In someembodiments the change to a native or genomic nucleic acid moleculeincludes but is not limited to: changes in the nucleic acid sequence dueto the degeneracy of the genetic code; codon optimization of the nucleicacid sequence for expression in plants; changes in the nucleic acidsequence to introduce at least one amino acid substitution, insertion,deletion and/or addition compared to the native or genomic sequence;removal of one or more intron associated with the genomic nucleic acidsequence; insertion of one or more heterologous introns; deletion of oneor more upstream or downstream regulatory regions associated with thegenomic nucleic acid sequence; insertion of one or more heterologousupstream or downstream regulatory regions; deletion of the 5′ and/or 3′untranslated region associated with the genomic nucleic acid sequence;insertion of a heterologous 5′ and/or 3′ untranslated region; andmodification of a polyadenylation site. In some embodiments thenon-genomic nucleic acid molecule is a cDNA. In some embodiments thenon-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

In some embodiments the polynucleotide encodes a PIP-45-1 polypeptidesufficiently homologous to the amino acid sequence of SEQ ID NO: 1, SEQID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27,SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO:39, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 234 or SEQ ID NO: 236 andwhich has insecticidal activity. “Sufficiently homologous” is usedherein to refer to an amino acid sequence that has at least about 50%,55%, 60%, 65%, 70%, or 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater sequence homology compared to areference sequence using one of the alignment programs described hereinusing standard parameters. One of skill in the art will recognize thatthese values can be appropriately adjusted to determine correspondinghomology of proteins taking into account amino acid similarity and thelike. As used herein the term “about” when used with sequence indentitymeans±0.5%. In some embodiments the sequence homology is against thefull length sequence of a PIP-45-1 polypeptide.

In some embodiments the polynucleotide encodes a PIP-45-1 polypeptidehaving at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequenceidentity compared to SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 19, SEQ IDNO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQID NO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45,SEQ ID NO: 234 or SEQ ID NO: 236.

In some embodiments the polynucleotide encodes a PIP-45-1 polypeptidehaving at least 99.1% or greater sequence identity compared to SEQ IDNO: 1. In some embodiments the polynucleotide encodes a PIP-45-1polypeptide having at least 99.4% or greater sequence identity comparedto SEQ ID NO: 17. In some embodiments the polynucleotide encodes aPIP-45-1 polypeptide having at least 99.6% or greater sequence identitycompared to SEQ ID NO: 19. In some embodiments the polynucleotideencodes a PIP-45-1 polypeptide having at least 87% or greater sequenceidentity compared to SEQ ID NO: 21. In some embodiments thepolynucleotide encodes a PIP-45-1 polypeptide having at least 88% orgreater sequence identity compared to SEQ ID NO: 23. In some embodimentsthe polynucleotide encodes a PIP-45-1 polypeptide having at least 99.1%or greater sequence identity compared to SEQ ID NO: 27. In someembodiments the polynucleotide encodes a PIP-45-1 polypeptide having atleast 99.8% or greater sequence identity compared to SEQ ID NO: 29. Insome embodiments the polynucleotide encodes a PIP-45-1 polypeptidehaving at least 92.3% or greater sequence identity compared to SEQ IDNO: 31. In some embodiments the polynucleotide encodes a PIP-45-1polypeptide having at least 91.1% or greater sequence identity comparedto SEQ ID NO: 33. In some embodiments the polynucleotide encodes aPIP-45-1 polypeptide having at least 95.4% or greater sequence identitycompared to SEQ ID NO: 35. In some embodiments the polynucleotideencodes a PIP-45-1 polypeptide having at least 93% or greater sequenceidentity compared to SEQ ID NO: 39. In some embodiments thepolynucleotide encodes a PIP-45-1 polypeptide having at least 97.5% orgreater sequence identity compared to SEQ ID NO: 43. In some embodimentsthe polynucleotide encodes a PIP-45-1 polypeptide having at least 70% orgreater sequence identity compared to SEQ ID NO: 45. In some embodimentsthe polynucleotide encodes a PIP-45-1 polypeptide having at least 94% orgreater sequence identity compared to SEQ ID NO: 234. In someembodiments the polynucleotide encodes a PIP-45-1 polypeptide having atleast 96% or greater sequence identity compared to SEQ ID NO: 236.

Polynucleotides encoding PIP-45-2 polypeptides are encompassed by thedisclosure. A variety of polynucleotides encoding PIP-45-2 polypeptidesare contemplated. One source of a polynucleotides encoding a PIP-45-2polypeptide or related protein is a bacterial strain that contains thepolynucleotide of SEQ ID NO: 109, SEQ ID NO: 125, SEQ ID NO: 127, SEQ IDNO: 129, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139,SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ IDNO: 153, SEQ ID NO: 221 or SEQ ID NO: 223 that encode the PIP-45-2polypeptide of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO:22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ IDNO: 34, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQID NO: 235 and SEQ ID NO: 237, respectively. One source of apolynucleotide encoding PIP-45-2 polypeptide or related protein is froma Pseudomonas, Thalassuspira, Paracoccusor Cellvibrio strain. One sourceof a polynucleotide encoding a PIP-45-2 polypeptide or related proteinsis from a Pseudomonas strain selected from but not limited toPseudomonas brenneri, Pseudomonas monteilii, Pseudomonas gessardii,Pseudomonas plecoglossicida, Pseudomonas putida, Pseudomonas poae,Pseudomonastrivialis, Pseudomonaslibanensis, Pseudomonas fluorescens andPseudomonas asplenii.

In some embodiments the nucleic acid molecule encoding the PIP-45-2polypeptide is a non-genomic nucleic acid sequence. In some embodimentsthe non-genomic nucleic acid molecule is a cDNA. In some embodiments thenon-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

In some embodiments the polynucleotide encodes a PIP-45-2 polypeptidesufficiently homologous to the amino acid sequence of SEQ ID NO: 2, SEQID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28,SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO:40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 235 or SEQ ID NO: 237 andwhich has insecticidal activity. “Sufficiently homologous” is usedherein to refer to an amino acid sequence that has at least about 50%,55%, 60%, 65%, 70%, or 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater sequence homology compared to areference sequence using one of the alignment programs described hereinusing standard parameters. One of skill in the art will recognize thatthese values can be appropriately adjusted to determine correspondinghomology of proteins taking into account amino acid similarity and thelike. In some embodiments the sequence homology is against the fulllength sequence of a PIP-45-2 polypeptide. In some embodiments thepolynucleotide encodes a PIP-45-2 polypeptide having at least about 50%,55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater sequence identity compared to SEQ IDNO: 2, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36,SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 235 or SEQ IDNO: 237.

In some embodiments the polynucleotide encodes a PIP-45-2 polypeptidehaving at least 99.2% or greater sequence identity compared to SEQ IDNO: 2. In some embodiments the polynucleotide encodes a PIP-45-2polypeptide having at least 98.5% or greater sequence identity comparedto SEQ ID NO: 18. In some embodiments the polynucleotide encodes aPIP-45-2 polypeptide having at least 96% or greater sequence identitycompared to SEQ ID NO: 20. In some embodiments the polynucleotideencodes a PIP-45-2 polypeptide having at least 80% or greater sequenceidentity compared to SEQ ID NO: 22. In some embodiments thepolynucleotide encodes a PIP-45-2 polypeptide having at least 81% orgreater sequence identity compared to SEQ ID NO: 24. In some embodimentsthe polynucleotide encodes a PIP-45-2 polypeptide having at least 99.5%or greater sequence identity compared to SEQ ID NO: 28. In someembodiments the polynucleotide encodes a PIP-45-2 polypeptide having atleast 98.5% or greater sequence identity compared to SEQ ID NO: 30. Insome embodiments the polynucleotide encodes a PIP-45-2 polypeptidehaving at least 92% or greater sequence identity compared to SEQ ID NO:32. In some embodiments the polynucleotide encodes a PIP-45-2polypeptide having at least 91.5% or greater sequence identity comparedto SEQ ID NO: 34. In some embodiments the polynucleotide encodes aPIP-45-2 polypeptide having at least 70% or greater sequence identitycompared to SEQ ID NO: 36. In some embodiments the polynucleotideencodes a PIP-45-2 polypeptide having at least 90% or greater sequenceidentity compared to SEQ ID NO: 40. In some embodiments thepolynucleotide encodes a PIP-45-2 polypeptide having at least 94% orgreater sequence identity compared to SEQ ID NO: 44. In some embodimentsthe polynucleotide encodes a PIP-45-2 polypeptide having at least 70% orgreater sequence identity compared to SEQ ID NO: 46. In some embodimentsthe polynucleotide encodes a PIP-45-2 polypeptide having at least 91% orgreater sequence identity compared to SEQ ID NO: 235. In someembodiments the polynucleotide encodes a PIP-45-2 polypeptide having atleast 93.5% or greater sequence identity compared to SEQ ID NO: 237.

Polynucleotides encoding PIP-64-1 polypeptides are encompassed by thedisclosure. A variety of polynucleotides encoding PIP-64-1 polypeptidesare contemplated. One source of a polynucleotide encoding a PIP-64-1polypeptide or related protein is a bacterial strain that contains thepolynucleotide of SEQ ID NO: 160, SEQ ID NO: 165 or SEQ ID NO: 224 thatencode the PIP-64-1 polypeptide of SEQ ID NO: 53, SEQ ID NO: 58 and SEQID NO: 238. One source of a polynucleotide encoding a PIP-64-1polypeptide or related protein is from a Pseudomonas, Enterobacter orAlcaligenes strain. One source of a polynucleotide encoding a PIP-64-1polypeptide or related proteins is from a Pseudomonas or Alcaligenesstrain selected from but not limited to Pseudomonas brenneri,Pseudomonas gessardii, Pseudomonas fluorescens, Pseudomonasbrassicacearum, Pseudomonas entomophila and Alcaligenes faecalis.

In some embodiments the nucleic acid molecule encoding the PIP-64-1polypeptide is a non-genomic nucleic acid sequence. In some embodimentsthe non-genomic nucleic acid molecule is a cDNA. In some embodiments thenon-genomic nucleic acid molecule is a synthetic nucleic acid sequence

In some embodiments the polynucleotide encodes a PIP-64-1 polypeptidesufficiently homologous to the amino acid sequence of SEQ ID NO: 53, SEQID NO: 58 or SEQ ID NO: 238 and which has insecticidal activity.“Sufficiently homologous” is used herein to refer to an amino acidsequence that has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greatersequence homology compared to a reference sequence using one of thealignment programs described herein using standard parameters. One ofskill in the art will recognize that these values can be appropriatelyadjusted to determine corresponding homology of proteins taking intoaccount amino acid similarity and the like. In some embodiments thesequence homology is against the full length sequence of a PIP-64-1polypeptide. In some embodiments the polynucleotide encodes a PIP-64-1polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to SEQ ID NO: 53, SEQ ID NO: 58 orSEQ ID NO: 238.

In some embodiments the polynucleotide encodes a PIP-64-1 polypeptidehaving at least 75% or greater sequence identity compared to SEQ ID NO:53. In some embodiments the polynucleotide encodes a PIP-64-1polypeptide having at least 99.7% or greater sequence identity comparedto SEQ ID NO: 58. In some embodiments the polynucleotide encodes aPIP-64-1 polypeptide having at least 75% or greater sequence identitycompared to SEQ ID NO: 238.

Polynucleotides encoding PIP-64-2 polypeptides are encompassed by thedisclosure. A variety of polynucleotides encodes a PIP-64-2 polypeptideare contemplated. One source of a polynucleotide encoding a PIP-64-2polypeptide or related protein is a bacterial strain that contains thepolynucleotide of SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 166 or SEQID NO: 225 that encode the PIP-64-2 polypeptide of SEQ ID NO: 54, SEQ IDNO: 55, SEQ ID NO: 59 and SEQ ID NO: 239, respectively. One source of apolynucleotide encoding a PIP-64-2 polypeptide or related protein isfrom a Pseudomonas, Enterobacter or Alcaligenes strain. One source of apolynucleotide encoding a PIP-64-2 polypeptide or related protein isfrom a Pseudomonas or Alcaligenes strain selected from but not limitedto Pseudomonas brenneri, Pseudomonas gessardii, Pseudomonas fluorescens,Pseudomonas brassicacearum, Pseudomonas entomophila and Alcaligenesfaecalis.

In some embodiments the nucleic acid molecule encoding the PIP-64-2polypeptide is a non-genomic nucleic acid sequence. In some embodimentsthe non-genomic nucleic acid molecule is a cDNA. In some embodiments thenon-genomic nucleic acid molecule is a synthetic nucleic acid sequence

In some embodiments the polynucleotide encodes a PIP-64-2 polypeptidesufficiently homologous to the amino acid sequence of SEQ ID NO: 54, SEQID NO: 55, SEQ ID NO: 59 or SEQ ID NO: 239 and which has insecticidalactivity. “Sufficiently homologous” is used herein to refer to an aminoacid sequence that has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence homology compared to a reference sequence using one ofthe alignment programs described herein using standard parameters. Oneof skill in the art will recognize that these values can beappropriately adjusted to determine corresponding homology of proteinstaking into account amino acid similarity and the like. In someembodiments the sequence homology is against the full length sequence ofa PIP-64-2 polypeptide. In some embodiments the PIP-64-2 polypeptide hasat least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identitycompared to SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 59 or SEQ ID NO:239.

In some embodiments the polynucleotide encodes a PIP-64-2 polypeptidehaving at least 70% or greater sequence identity compared to SEQ ID NO:54. In some embodiments the polynucleotide encodes a PIP-64-2polypeptide having at least 70% or greater sequence identity compared toSEQ ID NO: 55. In some embodiments the polynucleotide encodes a PIP-64-2polypeptide having at least 91% or greater sequence identity compared toSEQ ID NO: 59. In some embodiments the polynucleotide encodes a PIP-64-2polypeptide having at least 70% or greater sequence identity compared toSEQ ID NO: 239.

Polynucleotides encoding PIP-74-1 polypeptides are encompassed by thedisclosure. A variety of polynucleotides encoding PIP-74-1 polypeptidesare contemplated. One source of a polynucleotide encoding a PIP-74-1polypeptide or related protein is a bacterial strain that contains thepolynucleotide of SEQ ID NO: 180, SEQ ID NO: 182 or SEQ ID NO: 184 thatencode the PIP-74-1 polypeptide of SEQ ID NO: 73, SEQ ID NO: 75 and SEQID NO: 77, respectively. One source of the polynucleotide encoding aPIP-74-1 polypeptide or related protein is from a Pseudomonas strain.One source of the polynucleotide encoding a PIP-74-1 polypeptide orrelated proteins is from a Pseudomonas strain selected from but notlimited to Pseudomonas rhodesiae and Pseudomonas orientalis.

In some embodiments the nucleic acid molecule encoding the PIP-74-1polypeptide is a non-genomic nucleic acid sequence. In some embodimentsthe non-genomic nucleic acid molecule is a cDNA. In some embodiments thenon-genomic nucleic acid molecule is a synthetic nucleic acid sequence

In some embodiments the polynucleotide encodes a PIP-74-1 polypeptidesufficiently homologous to the amino acid sequence of SEQ ID NO: 73, SEQID NO: 75 or SEQ ID NO: 77 and which has insecticidal activity.“Sufficiently homologous” is used herein to refer to an amino acidsequence that has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greatersequence homology compared to a reference sequence using one of thealignment programs described herein using standard parameters. One ofskill in the art will recognize that these values can be appropriatelyadjusted to determine corresponding homology of proteins taking intoaccount amino acid similarity and the like. In some embodiments thesequence homology is against the full length sequence of a PIP-74-1polypeptide. In some embodiments the polynucleotide encodes a PIP-74-1polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to SEQ ID NO: 73, SEQ ID NO: 75 orSEQ ID NO: 77.

In some embodiments the polynucleotide encodes a PIP-74-1 polypeptidehaving at least 75% or greater sequence identity compared to SEQ ID NO:73. In some embodiments the polynucleotide encodes a PIP-74-1polypeptide having at least 75% or greater sequence identity compared toSEQ ID NO: 75. In some embodiments the polynucleotide encodes a PIP-74-1polypeptide having at least 75% or greater sequence identity compared toSEQ ID NO: 77.

Polynucleotides encoding PIP-74-2 polypeptides are encompassed by thedisclosure. A variety of polynucleotides encoding PIP-74-2 polypeptidesare contemplated. One source of the polynucleotide encoding a PIP-74-2polypeptide or related protein is a bacterial strain that contains thepolynucleotide of SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185 thatencode the PIP-74-2 polypeptide of SEQ ID NO: 74, SEQ ID NO: 76 and SEQID NO: 78, respectively. One source of the polynucleotide encoding aPIP-74-2 polypeptide or related proteins is from a Pseudomonas strain.One source of a PIP-74-2 polypeptide or related proteins is from aPseudomonas strain selected from but not limited to Pseudomonasrhodesiae and Pseudomonas orientalis.

In some embodiments the nucleic acid molecule encoding the PIP-74-2polypeptide is a non-genomic nucleic acid sequence. In some embodimentsthe non-genomic nucleic acid molecule is a cDNA. In some embodiments thenon-genomic nucleic acid molecule is a synthetic nucleic acid sequence

In some embodiments the polynucleotide encodes a PIP-74-2 polypeptidesufficiently homologous to the amino acid sequence of SEQ ID NO: 74, SEQID NO: 76 or SEQ ID NO: 78 and which has insecticidal activity.“Sufficiently homologous” is used herein to refer to an amino acidsequence that has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greatersequence homology compared to a reference sequence using one of thealignment programs described herein using standard parameters. One ofskill in the art will recognize that these values can be appropriatelyadjusted to determine corresponding homology of proteins taking intoaccount amino acid similarity and the like. In some embodiments thesequence homology is against the full length sequence of a PIP-74-2polypeptide. In some embodiments the polynucleotide encodes a PIP-74-2polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to SEQ ID NO: 74, SEQ ID NO: 76 orSEQ ID NO: 78.

In some embodiments the polynucleotide encodes a PIP-74-2 polypeptidehaving at least 75% or greater sequence identity compared to SEQ ID NO:74. In some embodiments the polynucleotide encodes a PIP-74-2polypeptide having at least 75% or greater sequence identity compared toSEQ ID NO: 76. In some embodiments the polynucleotide encodes a PIP-74-2polypeptide having at least 75% or greater sequence identity compared toSEQ ID NO: 78.

Polynucleotides encoding PIP-75 polypeptides are encompassed by thedisclosure. A variety of polynucleotides encoding a PIP-75 polypeptideare contemplated. One source of a polynucleotide encoding a PIP-75polypeptide or related protein is a bacterial strain that contains thepolynucleotide of SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ IDNO: 191, SEQ ID NO: 192, SEQ ID NO: 193 or SEQ ID NO: 194 that encodethe PIP-75 polypeptide of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81,SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87. Onesource of a polynucleotide encoding a PIP-75 polypeptide or relatedprotein is from a Pseudomonas, Enterobacter or Serratia strain. Onesource of a PIP-75 polypeptide or related proteins is from aPseudomonas, Enterobacter or Serratia strain selected from but notlimited to Pseudomonas antarctica, Pseudomonas orientalis, Enterobacterasburiae, Serratia plymuthica, and Serratia liquefaciens.

In some embodiments the nucleic acid molecule encoding the PIP-75polypeptide is a non-genomic nucleic acid sequence. In some embodimentsthe non-genomic nucleic acid molecule is a cDNA. In some embodiments thenon-genomic nucleic acid molecule is a synthetic nucleic acid sequence

In some embodiments the polynucleotide encodes a PIP-75 polypeptidesufficiently homologous to the amino acid sequence of SEQ ID NO: 79, SEQID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 orSEQ ID NO: 87 and which has insecticidal activity. “Sufficientlyhomologous” is used herein to refer to an amino acid sequence that hasat least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homologycompared to a reference sequence using one of the alignment programsdescribed herein using standard parameters. One of skill in the art willrecognize that these values can be appropriately adjusted to determinecorresponding homology of proteins taking into account amino acidsimilarity and the like. In some embodiments the sequence homology isagainst the full length sequence of a PIP-75 polypeptide. In someembodiments the polynucleotide encodes a PIP-75 polypeptide having atleast about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identitycompared to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84,SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87.

In some embodiments the polynucleotide encodes a PIP-75 polypeptidehaving at least 75% or greater sequence identity compared to SEQ ID NO:79. In some embodiments the polynucleotide encodes a PIP-75 polypeptidehaving at least 75% or greater sequence identity compared to SEQ ID NO:80. In some embodiments the polynucleotide encodes a PIP-75 polypeptidehaving at least 86% or greater sequence identity compared to SEQ ID NO:81. In some embodiments the polynucleotide encodes a PIP-75 polypeptidehaving at least 75% or greater sequence identity compared to SEQ ID NO:84. In some embodiments the polynucleotide encodes a PIP-75 polypeptidehaving at least 75% or greater sequence identity compared to SEQ ID NO:85. In some embodiments the polynucleotide encodes a PIP-75 polypeptidehaving at least 75% or greater sequence identity compared to SEQ ID NO:86. In some embodiments the polynucleotide encodes a PIP-75 polypeptidehaving at least 75% or greater sequence identity compared to SEQ ID NO:87.

Polynucleotides encoding PIP-77 polypeptides are encompassed by thedisclosure. A variety of polynucleotides encoding a PIP-77 polypeptideare contemplated. One source of a polynucleotide encoding a PIP-77polypeptide or related proteins is a bacterial strain that contains thepolynucleotide of SEQ ID NO: 195, SEQ ID NO:196, SEQ ID NO:197, SEQ IDNO:198, SEQ ID NO:199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202,SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 207, SEQ IDNO: 227, SEQ ID NO: 228 or SEQ ID NO: 231 that encode the PIP-77polypeptide of SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO:92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ IDNO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 241, SEQ ID NO: 242and SEQ ID NO: 245, respectively. One source of a polynucleotideencoding a PIP-77 polypeptide or related proteins is from a Pseudomonas,Enterobacter, Shewanella, Haemophilus or Aeromonas strain. One source ofa PIP-77 polypeptide or related proteins is from a Pseudomonas strainselected from but not limited to Pseudomonas chlororaphis, Pseudomonasbrassicacearum, Pseudomonas fluorescens and Pseudomonas rhodesiae.

In some embodiments the nucleic acid molecule encoding the PIP-77polypeptide is a non-genomic nucleic acid sequence. In some embodimentsthe non-genomic nucleic acid molecule is a cDNA. In some embodiments thenon-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

In some embodiments the polynucleotide encodes a PIP-77 polypeptidesufficiently homologous to the amino acid sequence of SEQ ID NO: 88, SEQID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94,SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO:100, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 245 and which hasinsecticidal activity. “Sufficiently homologous” is used herein to referto an amino acid sequence that has at least about 50%, 55%, 60%, 65%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater sequence homology compared to a reference sequenceusing one of the alignment programs described herein using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding homology ofproteins taking into account amino acid similarity and the like. In someembodiments the sequence homology is against the full length sequence ofa PIP-77 polypeptide. In some embodiments the polynucleotide encodes aPIP-77 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%,72%, 73%, 74cY0, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to SEQ ID NO: 88, SEQ ID NO: 89, SEQID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95,SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO:241, SEQ ID NO: 242 or SEQ ID NO: 245.

In some embodiments the polynucleotide encodes a PIP-77 polypeptidehaving at least 93% or greater sequence identity compared to SEQ ID NO:88. In some embodiments the polynucleotide encodes a PIP-77 polypeptidehaving at least 97% or greater sequence identity compared to SEQ ID NO:89. In some embodiments the polynucleotide encodes a PIP-77 polypeptidehaving at least 99% or greater sequence identity compared to SEQ ID NO:90. In some embodiments the polynucleotide encodes a PIP-77 polypeptidehaving at least 97% or greater sequence identity compared to SEQ ID NO:92. In some embodiments the polynucleotide encodes a PIP-77 polypeptidehaving at least 87% or greater sequence identity compared to SEQ ID NO:93. In some embodiments the polynucleotide encodes a PIP-77 polypeptidehaving at least 86% or greater sequence identity compared to SEQ ID NO:94. In some embodiments the polynucleotide encodes a PIP-77 polypeptidehaving at least 85% or greater sequence identity compared to SEQ ID NO:95. In some embodiments the polynucleotide encodes a PIP-77 polypeptidehaving at least 84% or greater sequence identity compared to SEQ ID NO:96. In some embodiments the polynucleotide encodes a PIP-77 polypeptidehaving at least 85% or greater sequence identity compared to SEQ ID NO:97. In some embodiments the polynucleotide encodes a PIP-77 polypeptidehaving at least 83% or greater sequence identity compared to SEQ ID NO:98. In some embodiments the polynucleotide encodes a PIP-77 polypeptidehaving at least 79% or greater sequence identity compared to SEQ ID NO:100.

These polynucleotide sequences were isolated from a Pseudomonas or otherbacterial host and are thus suitable for expression of the encodedinsecticidal polypeptides in other bacterial hosts that include but arenot limited to Agrobacterium, Bacillus, Escherichia, Salmonella,Pseudomonas and Rhizobium bacterial host cells. The polynucleotides arealso useful as probes for isolating homologous or substantiallyhomologous polynucleotides that encode the insecticidal polypeptides ofthe disclosure or related proteins. Such probes can be used to identifyhomologous or substantially homologous polynucleotides derived fromPseudomonas or other related bacteria.

Polynucleotides that encode an insecticidal polypeptide can also besynthesized de novo from a polypeptide sequence. The sequence of thepolynucleotide gene can be deduced from a polypeptide sequence throughuse of the genetic code. Computer programs such as “BackTranslate” (GCG™Package, Acclerys, Inc. San Diego, Calif.) can be used to convert apeptide sequence to the corresponding nucleotide sequence encoding thepeptide. Furthermore, synthetic polynucleotide sequences of thedisclosure can be designed so that they will be expressed in plants.U.S. Pat. No. 5,500,365 describes a method for synthesizing plant genesto improve the expression level of the protein encoded by thesynthesized gene. This method relates to the modification of thestructural gene sequences of the exogenous transgene, to cause them tobe more efficiently transcribed, processed, translated and expressed bythe plant. Features of genes that are expressed well in plants includeelimination of sequences that can cause undesired intron splicing orpolyadenylation in the coding region of a gene transcript whileretaining substantially the amino acid sequence of the toxic portion ofthe insecticidal protein. A similar method for obtaining enhancedexpression of transgenes in monocotyledonous plants is disclosed in U.S.Pat. No. 5,689,052.

“Complement” is used herein to refer to a nucleic acid sequence that issufficiently complementary to a given nucleic acid sequence such that itcan hybridize to the given nucleic acid sequence to thereby form astable duplex. “Polynucleotide sequence variants” is used herein torefer to a nucleic acid sequence that except for the degeneracy of thegenetic code encodes the same polypeptide.

In some embodiments a nucleic acid molecule encoding the insecticidalpolypeptide of the disclosure is a non-genomic nucleic acid sequence. Asused herein a “non-genomic nucleic acid sequence” or “non-genomicnucleic acid molecule” refers to a nucleic acid molecule that has one ormore change in the nucleic acid sequence compared to a native or genomicnucleic acid sequence. In some embodiments the change to a native orgenomic nucleic acid molecule includes but is not limited to: changes inthe nucleic acid sequence due to the degeneracy of the genetic code;codon optimization of the nucleic acid sequence for expression inplants; changes in the nucleic acid sequence to introduce at least oneamino acid substitution, insertion, deletion and/or addition compared tothe native or genomic sequence; removal of one or more intron associatedwith the genomic nucleic acid sequence; insertion of one or moreheterologous introns; deletion of one or more upstream or downstreamregulatory regions associated with the genomic nucleic acid sequence;insertion of one or more heterologous upstream or downstream regulatoryregions; deletion of the 5′ and/or 3′ untranslated region associatedwith the genomic nucleic acid sequence; insertion of a heterologous 5′and/or 3′ untranslated region; and modification of a polyadenylationsite. In some embodiments the non-genomic nucleic acid molecule is acDNA.

Also provided are nucleic acid molecules that encode transcriptionand/or translation products that are subsequently spliced to ultimatelyproduce functional insecticidal polypeptides. Splicing can beaccomplished in vitro or in vivo, and can involve cis- ortrans-splicing. The substrate for splicing can be polynucleotides (e.g.,RNA transcripts) or polypeptides. An example of cis-splicing of apolynucleotide is where an intron inserted into a coding sequence isremoved and the two flanking exon regions are spliced to generate ainsecticidal polypeptide encoding sequence of the disclosure. An exampleof trans splicing would be where a polynucleotide is encrypted byseparating the coding sequence into two or more fragments that can beseparately transcribed and then spliced to form the full-lengthpesticidal encoding sequence. The use of a splicing enhancer sequence,which can be introduced into a construct, can facilitate splicing eitherin cis or trans-splicing of polypeptides (U.S. Pat. Nos. 6,365,377 and6,531,316). Thus, in some embodiments the polynucleotides do notdirectly encode a full-length insecticidal polypeptide of thedisclsosure, but rather encode a fragment or fragments of aninsecticidal polypeptide of the disclsosure. These polynucleotides canbe used to express a functional Insecticidal polypeptide of thedisclosure through a mechanism involving splicing, where splicing canoccur at the level of polynucleotide (e.g., intron/exon) and/orpolypeptide (e.g., intein/extein). This can be useful, for example, incontrolling expression of pesticidal activity, since a functionalpesticidal polypeptide will only be expressed if all required fragmentsare expressed in an environment that permits splicing processes togenerate functional product. In another example, introduction of one ormore insertion sequences into a polynucleotide can facilitaterecombination with a low homology polynucleotide; use of an intron orintein for the insertion sequence facilitates the removal of theintervening sequence, thereby restoring function of the encoded variant.

Nucleic acid molecules that are fragments of these nucleic acidsequences encoding insecticidal polypeptides are also encompassed by theembodiments. “Fragment” as used herein refers to a portion of thenucleic acid sequence encoding an insecticidal polypeptide of thedisclsosure. A fragment of a nucleic acid sequence may encode abiologically active portion of an insecticidal polypeptide of thedisclsosure or it may be a fragment that can be used as a hybridizationprobe or PCR primer using methods disclosed below. Nucleic acidmolecules that are fragments of a nucleic acid sequence encoding aninsecticidal polypeptide of the disclsosure comprise at least about 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or 260,contiguous nucleotides or up to the number of nucleotides present in afull-length nucleic acid sequence encoding an insecticidal polypeptideof the disclsosure disclosed herein, depending upon the intended use.“Contiguous nucleotides” is used herein to refer to nucleotide residuesthat are immediately adjacent to one another. Fragments of the nucleicacid sequences of the embodiments will encode protein fragments thatretain the biological activity of the insecticidal polypeptide of thedisclosure and, hence, retain insecticidal activity. “Retainsinsecticidal activity” is used herein to refer to a polypeptide havingat least about 10%, at least about 30%, at least about 50%, at leastabout 70%, 80%, 90%, 95% or higher of the insecticidal activity of thefull-length native polypeptide. In one embodiment, the insecticidalactivity is Lepidoptera activity. In one embodiment, the insecticidalactivity is against a Coleopteran species. In one embodiment, theinsecticidal activity is against a Diabrotica species. In oneembodiment, the insecticidal activity is against one or more insectpests of the corn rootworm complex: Western corn rootworm, Diabroticavirgifera virgifera; northern corn rootworm, D. barberi: Southern cornrootworm or spotted cucumber beetle; Diabrotica undecimpunctata howardi,and the Mexican corn rootworm, D. virgifera zeae. In one embodiment, theinsecticidal activity is against Western corn rootworm, Diabroticavirgifera virgifera.

In some embodiments a fragment of a nucleic acid sequence encoding aninsecticidal polypeptide of the disclsosure encoding a biologicallyactive portion of a protein will encode at least about 15, 20, 30, 40,50, 60, 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 or 85, contiguousamino acids or up to the total number of amino acids present in afull-length insecticidal polypeptide of the embodiments. In someembodiments, the fragment is an N-terminal and/or a C-terminaltruncation of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14 or more amino acids from the N-terminus and/or C-terminus byproteolysis, insertion of a start codon, deletion of the codons encodingthe deleted amino acids with the concomitant insertion of a stop codonor by insertion of a stop codon in the coding sequence.

The present disclosure provides isolated or recombinant polynucleotidesthat encode any of the insecticidal polypeptides disclosed herein. Thosehaving ordinary skill in the art will readily appreciate that due to thedegeneracy of the genetic code, a multitude of nucleotide sequencesencoding insecticidal polypeptides of the present disclosure exist.Table 1 is a codon table that provides the synonymous codons for eachamino acid. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU allencode the amino acid arginine. Thus, at every position in the nucleicacids of the disclosure where an arginine is specified by a codon, thecodon can be altered to any of the corresponding codons described abovewithout altering the encoded polypeptide. It is understood that U in anRNA sequence corresponds to T in a DNA sequence.

TABLE 1 Alanine Ala GCA GCC GCG GCU Cysteine Cys UGC UGU Aspartic acidAsp GAC GAU Glutamic acid Glu GAA GAG Phenylalanine Phe UUC UUU GlycineGly GGA GGC GGG GGU Histidine His CAC CAU Isoleucine Ile AUA AUC AUULysine Lys AAA AAG Leucine Leu UUA UUG CUA CUC CUG CUU Methionine MetAUG Asparagine Asn AAC AAU Proline Pro CCA CCC CCG CCU Glutamine GlnCAA CAG Arginine Arg AGA AGG CGA CGC CGG CGU Serine SerAGC AGU UCA UCC UCG UCU T reonine Thr ACA ACC ACG ACU Valine ValGUA GUC GUG UU Tryptophan Trp UGG Tyrosine Tyr UAC UAU

The skilled artisan will further appreciate that changes can beintroduced by mutation of the nucleic acid sequences thereby leading tochanges in the amino acid sequence of the encoded insecticidalpolypeptides, without altering the biological activity of the proteins.Thus, variant nucleic acid molecules can be created by introducing oneor more nucleotide substitutions, additions and/or deletions into thecorresponding nucleic acid sequence disclosed herein, such that one ormore amino acid substitutions, additions or deletions are introducedinto the encoded protein. Mutations can be introduced by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleic acid sequences are also encompassed bythe present disclosure.

Alternatively, variant nucleic acid sequences can be made by introducingmutations randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forability to confer pesticidal activity to identify mutants that retainactivity. Following mutagenesis, the encoded protein can be expressedrecombinantly, and the activity of the protein can be determined usingstandard assay techniques.

The polynucleotides of the disclosure and fragments thereof areoptionally used as substrates for a variety of recombination andrecursive recombination reactions, in addition to standard cloningmethods as set forth in, e.g., Ausubel, Berger and Sambrook, i.e., toproduce additional pesticidal polypeptide homologues and fragmentsthereof with desired properties. A variety of such reactions are known,including those developed by the inventors and their co-workers. Methodsfor producing a variant of any nucleic acid listed herein comprisingrecursively recombining such polynucleotide with a second (or more)polynucleotide, thus forming a library of variant polynucleotides arealso embodiments of the disclosure, as are the libraries produced, thecells comprising the libraries and any recombinant polynucleotideproduces by such methods. Additionally, such methods optionally compriseselecting a variant polynucleotide from such libraries based onpesticidal activity, as is wherein such recursive recombination is donein vitro or in vivo.

A variety of diversity generating protocols, including nucleic acidrecursive recombination protocols are available and fully described inthe art. The procedures can be used separately, and/or in combination toproduce one or more variants of a nucleic acid or set of nucleic acids,as well as variants of encoded proteins. Individually and collectively,these procedures provide robust, widely applicable ways of generatingdiversified nucleic acids and sets of nucleic acids (including, e.g.,nucleic acid libraries) useful, e.g., for the engineering or rapidevolution of nucleic acids, proteins, pathways, cells and/or organismswith new and/or improved characteristics.

While distinctions and classifications are made in the course of theensuing discussion for clarity, it will be appreciated that thetechniques are often not mutually exclusive. Indeed, the various methodscan be used singly or in combination, in parallel or in series, toaccess diverse sequence variants.

The result of any of the diversity generating procedures describedherein can be the generation of one or more nucleic acids, which can beselected or screened for nucleic acids with or which confer desirableproperties or that encode proteins with or which confer desirableproperties. Following diversification by one or more of the methodsherein or otherwise available to one of skill, any nucleic acids thatare produced can be selected for a desired activity or property, e.g.pesticidal activity or, such activity at a desired pH, etc. This caninclude identifying any activity that can be detected, for example, inan automated or automatable format, by any of the assays in the art,see, e.g., discussion of screening of insecticidal activity, infra. Avariety of related (or even unrelated) properties can be evaluated, inserial or in parallel, at the discretion of the practitioner.

Descriptions of a variety of diversity generating procedures forgenerating modified nucleic acid sequences, e.g., those coding forpolypeptides having pesticidal activity or fragments thereof, are foundin the following publications and the references cited therein: Soong,et al., (2000) Nat Genet 25(4):436-439; Stemmer, et al., (1999) TumorTargeting 4:1-4; Ness, et al., (1999) Nat Biotechnol 17:893-896; Chang,et al., (1999) Nat Biotechnol 17:793-797; Minshull and Stemmer, (1999)Curr Opin Chem Biol 3:284-290; Christians, et al., (1999) Nat Biotechnol17:259-264; Crameri, et al., (1998) Nature 391:288-291; Crameri, et al.,(1997) Nat Biotechnol 15:436-438; Zhang, et al., (1997) PNAS USA94:4504-4509; Patten, et al., (1997) Curr Opin Biotechnol 8:724-733;Crameri, et al., (1996) Nat Med 2:100-103; Crameri, et al., (1996) NatBiotechnol 14:315-319; Gates, et al., (1996) J Mol Biol 255:373-386;Stemmer, (1996) “Sexual PCR and Assembly PCR” In: The Encyclopedia ofMolecular Biology. VCH Publishers, New York. pp. 447-457; Crameri andStemmer, (1995) BioTechniques 18:194-195; Stemmer, et al., (1995) Gene,164:49-53; Stemmer, (1995) Science 270: 1510; Stemmer, (1995)Bio/Technology 13:549-553; Stemmer, (1994) Nature 370:389-391 andStemmer, (1994) PNAS USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling, et al., (1997) Anal Biochem254(2):157-178; Dale, et al., (1996) Methods Mol Biol 57:369-374; Smith,(1985) Ann Rev Genet 19:423-462; Botstein and Shortle, (1985) Science229:1193-1201; Carter, (1986) Biochem J 237:1-7 and Kunkel, (1987) “Theefficiency of oligonucleotide directed mutagenesis” in Nucleic Acids &Molecular Biology (Eckstein and Lilley, eds., Springer Verlag, Berlin));mutagenesis using uracil containing templates (Kunkel, (1985) PNAS USA82:488-492; Kunkel, et al., (1987) Methods Enzymol 154:367-382 and Bass,et al., (1988) Science 242:240-245); oligonucleotide-directedmutagenesis (Zoller and Smith, (1983) Methods Enzymol 100:468-500;Zoller and Smith, (1987) Methods Enzymol 154:329-350 (1987); Zoller andSmith, (1982) Nucleic Acids Res 10:6487-6500), phosphorothioate-modifiedDNA mutagenesis (Taylor, et al., (1985) Nucl Acids Res 13:8749-8764;Taylor, et al., (1985) Nucl Acids Res 13:8765-8787 (1985); Nakamaye andEckstein, (1986) Nucl Acids Res 14:9679-9698; Sayers, et al., (1988)Nucl Acids Res 16:791-802 and Sayers, et al., (1988) Nucl Acids Res16:803-814); mutagenesis using gapped duplex DNA (Kramer, et al., (1984)Nucl Acids Res 12:9441-9456; Kramer and Fritz, (1987) Methods Enzymol154:350-367; Kramer, et al., (1988) Nucl Acids Res 16:7207 and Fritz, etal., (1988) Nucl Acids Res 16:6987-6999).

Additional suitable methods include point mismatch repair (Kramer, etal., (1984) Cell 38:879-887), mutagenesis using repair-deficient hoststrains (Carter, et al., (1985) Nucl Acids Res 13:4431-4443 and Carter,(1987) Methods in Enzymol 154:382-403), deletion mutagenesis(Eghtedarzadeh and Henikoff, (1986) Nucl Acids Res 14:5115),restriction-selection and restriction-purification (Wells, et al.,(1986) Phil Trans R Soc Lond A 317:415-423), mutagenesis by total genesynthesis (Nambiar, et al., (1984) Science 223:1299-1301; Sakamar andKhorana, (1988) Nucl Acids Res 14:6361-6372; Wells, et al., (1985) Gene34:315-323 and Grundstrom, et al., (1985) Nucl Acids Res 13:3305-3316),double-strand break repair (Mandecki, (1986) PNAS USA, 83:7177-7181 andArnold, (1993) Curr Opin Biotech 4:450-455). Additional details on manyof the above methods can be found in Methods Enzymol Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Additional details regarding various diversity generating methods can befound in the following US Patents, PCT Publications and Applications andEPO publications: U.S. Pat. Nos. 5,723,323, 5,763,192, 5,814,476,5,817,483, 5,824,514, 5,976,862, 5,605,793, 5,811,238, 5,830,721,5,834,252, 5,837,458, WO 1995/22625, WO 1996/33207, WO 1997/20078, WO1997/35966, WO 1999/41402, WO 1999/41383, WO 1999/41369, WO 1999/41368,EP 752008, EP 0932670, WO 1999/23107, WO 1999/21979, WO 1998/31837, WO1998/27230, WO 1998/27230, WO 2000/00632, WO 2000/09679, WO 1998/42832,WO 1999/29902, WO 1998/41653, WO 1998/41622, WO 1998/42727, WO2000/18906, WO 2000/04190, WO 2000/42561, WO 2000/42559, WO 2000/42560,WO 2001/23401 and PCT/US01/06775.

The nucleotide sequences of the embodiments can also be used to isolatecorresponding sequences from other organisms, particularly otherbacteria, particularly a Pseudomonas species and more particularly aPseudomonas putida, a Pseudomonas fulva or a Pseudomonas chlororaphisstrain. In this manner, methods such as PCR, hybridization, and the likecan be used to identify such sequences based on their sequence homologyto the sequences set forth herein. Sequences that are selected based ontheir sequence identity to the entire sequences set forth herein or tofragments thereof are encompassed by the embodiments. Such sequencesinclude sequences that are orthologs of the disclosed sequences. Theterm “orthologs” refers to genes derived from a common ancestral geneand which are found in different species as a result of speciation.Genes found in different species are considered orthologs when theirnucleotide sequences and/or their encoded protein sequences sharesubstantial identity as defined elsewhere herein. Functions of orthologsare often highly conserved among species.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook, et al., (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.), hereinafter “Sambrook”. See also, Innis, et al, eds.(1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Known methods of PCR include,but are not limited to, methods using paired primers, nested primers,single specific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

To identify potential insecticidal polypeptides from bacterialcollections, the bacterial cell lysates can be screened with antibodiesgenerated against an insecticidal polypeptide of the disclosure usingWestern blotting and/or ELISA methods. This type of assays can beperformed in a high throughput fashion. Positive samples can be furtheranalyzed by various techniques such as antibody based proteinpurification and identification. Methods of generating antibodies arewell known in the art as discussed infra.

Alternatively, mass spectrometry based protein identification method canbe used to identify homologs of the insecticidal polypeptides usingprotocols in the literatures (Scott Patterson, (1998), 10.22, 1-24,Current Protocol in Molecular Biology published by John Wiley & SonInc). Specifically, LC-MS/MS based protein identification method is usedto associate the MS data of given cell lysate or desired molecularweight enriched samples (excised from SDS-PAGE gel of relevant molecularweight bands) with sequence information of the insecticidal polypeptidesof the disclosure. Any match in peptide sequences indicates thepotential of having the homologs in the samples. Additional techniques(protein purification and molecular biology) can be used to isolate theprotein and identify the sequences of the homologs.

In hybridization methods, all or part of the pesticidal nucleic acidsequence can be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook and Russell, (2001), supra. Theso-called hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments or other oligonucleotides and may be labeledwith a detectable group such as 32P or any other detectable marker, suchas other radioisotopes, a fluorescent compound, an enzyme or an enzymeco-factor. Probes for hybridization can be made by labeling syntheticoligonucleotides based on the insecticidal polypeptide-encoding nucleicacid sequence disclosed herein. Degenerate primers designed on the basisof conserved nucleotides or amino acid residues in the nucleic acidsequence or encoded amino acid sequence can additionally be used. Theprobe typically comprises a region of nucleic acid sequence thathybridizes under stringent conditions to at least about 12, at leastabout 25, at least about 50, 75, 100, 125, 150, 175 or 200 consecutivenucleotides of nucleic acid sequence encoding an insecticidalpolypeptide of the disclosure or a fragment or variant thereof. Methodsfor the preparation of probes for hybridization are generally known inthe art and are disclosed in Sambrook and Russell, (2001), supra, hereinincorporated by reference.

For example, an entire nucleic acid sequence, encoding an insecticidalpolypeptide of the disclosure, disclosed herein or one or more portionsthereof may be used as a probe capable of specifically hybridizing tocorresponding nucleic acid sequences encoding insecticidalpolypeptide-like sequences and messenger RNAs. To achieve specifichybridization under a variety of conditions, such probes includesequences that are unique and are preferably at least about 10nucleotides in length or at least about 20 nucleotides in length. Suchprobes may be used to amplify corresponding pesticidal sequences from achosen organism by PCR. This technique may be used to isolate additionalcoding sequences from a desired organism or as a diagnostic assay todetermine the presence of coding sequences in an organism. Hybridizationtechniques include hybridization screening of plated DNA libraries(either plaques or colonies; see, for example, Sambrook, et al., (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. “Stringent conditions” or “stringent hybridizationconditions” is used herein to refer to conditions under which a probewill hybridize to its target sequence to a detectably greater degreethan to other sequences (e.g., at least 2-fold over background).Stringent conditions are sequence-dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences that are 100%complementary to the probe can be identified (homologous probing).Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, preferably less than 500 nucleotides inlength.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the Tm can be approximated from theequation of Meinkoth and Wahl, (1984) Anal. Biochem. 138:267-284:Tm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is themolarity of monovalent cations, % GC is the percentage of guanosine andcytosine nucleotides in the DNA, % form is the percentage of formamidein the hybridization solution, and L is the length of the hybrid in basepairs. The Tm is the temperature (under defined ionic strength and pH)at which 50% of a complementary target sequence hybridizes to aperfectly matched probe. Tm is reduced by about 1° C. for each 1% ofmismatching; thus, Tm, hybridization, and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with 90% identity are sought, the Tm can be decreased 10°C. Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3or 4° C. lower than the thermal melting point (Tm); moderately stringentconditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10°C. lower than the thermal melting point (Tm); low stringency conditionscan utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or 20° C.lower than the thermal melting point (Tm). Using the equation,hybridization and wash compositions, and desired Tm, those of ordinaryskill will understand that variations in the stringency of hybridizationand/or wash solutions are inherently described. If the desired degree ofmismatching results in a Tm of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen, (1993)Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, N.Y.); and Ausubel, et al., eds. (1995) Current Protocols inMolecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience,New York). See, Sambrook, et al., (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.).

Proteins and Variants and Fragments Thereof

One aspect of the disclosure is isolated insecticidal polypeptides.

PIP-45-1 polypeptides are encompassed by the disclosure. “PseudomonasInsecticidal Protein-45-1”, “PIP-45-1 polypeptide” or “PIP-45-1 protein”as used herein interchangeably refers to a polypeptide havinginsecticidal activity against one or more insect pests of theLepidoptera and/or Coleoptera orders, and is sufficiently homologous tothe protein of SEQ ID NO: 1. A variety of PIP-45-1 polypeptides arecontemplated. One source of a PIP-45-1 polypeptide or related proteinsis a bacterial strain that contains the polynucleotide of SEQ ID NO:108, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 220 orSEQ ID NO: 222 that encode the PIP-45-1 polypeptide of SEQ ID NO: 1, SEQID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27,SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO:39, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 234 and SEQ ID NO: 236,respectively. One source of a PIP-45-1 polypeptide or related proteinsis from a Pseudomonas, Thalassuspira, Paracoccus or Cellvibrio strain.One source of a PIP-45-1 polypeptide or related proteins is from aPseudomonas strain selected from but not limited to Pseudomonasbrenneri, Pseudomonas monteilii, Pseudomonas gessardii, Pseudomonasplecoglossicida, Pseudomonas putida, Pseudomonas poae, Pseudomonastrivialis, Pseudomonas libanensis, Pseudomonas fluorescens andPseudomonas asplenii.

In some embodiments a PIP-45-1 polypeptide is sufficiently homologous tothe amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 19,SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ IDNO: 45, SEQ ID NO: 234 or SEQ ID NO: 236 and has insecticidal activity.“Sufficiently homologous” is used herein to refer to an amino acidsequence that has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greatersequence homology compared to a reference sequence using one of thealignment programs described herein using standard parameters. One ofskill in the art will recognize that these values can be appropriatelyadjusted to determine corresponding homology of proteins taking intoaccount amino acid similarity and the like. In some embodiments thesequence homology is against the full length sequence of a PIP-45-1polypeptide.

In some embodiments the PIP-45-1 polypeptide has at least about 50%,55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%or greater sequence identity compared to SEQ IDNO: 1, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35,SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 234 or SEQ IDNO: 236.

In some embodiments the PIP-45-1 polypeptide has at least 99.1% orgreater sequence identity compared to SEQ ID NO: 1. In some embodimentsthe PIP-45-1 polypeptide has at least 99.4% or greater sequence identitycompared to SEQ ID NO: 17. In some embodiments the PIP-45-1 polypeptidehas at least 99.6% or greater sequence identity compared to SEQ ID NO:19. In some embodiments the PIP-45-1 polypeptide has at least 87% orgreater sequence identity compared to SEQ ID NO: 21. In some embodimentsthe PIP-45-1 polypeptide has at least 88% or greater sequence identitycompared to SEQ ID NO: 23. In some embodiments the PIP-45-1 polypeptidehas at least 99.1% or greater sequence identity compared to SEQ ID NO:27. In some embodiments the PIP-45-1 polypeptide has at least 99.8% orgreater sequence identity compared to SEQ ID NO: 29. In some embodimentsthe PIP-45-1 polypeptide has at least 92.3% or greater sequence identitycompared to SEQ ID NO: 31. In some embodiments the PIP-45-1 polypeptidehas at least 91.1% or greater sequence identity compared to SEQ ID NO:33. In some embodiments the PIP-45-1 polypeptide has at least 95.4% orgreater sequence identity compared to SEQ ID NO: 35. In some embodimentsthe PIP-45-1 polypeptide has at least 93% or greater sequence identitycompared to SEQ ID NO: 39. In some embodiments the PIP-45-1 polypeptidehas at least 97.5% or greater sequence identity compared to SEQ ID NO:43. In some embodiments the PIP-45-1 polypeptide has at least 70% orgreater sequence identity compared to SEQ ID NO: 45.

PIP-45-2 polypeptides are encompassed by the disclosure. “PseudomonasInsecticidal Protein-45-2”, “PIP-45-2 polypeptide” or “PIP-45-2 protein”as used herein interchangeably refers to a polypeptide havinginsecticidal activity against one or more insect pests of theLepidoptera and/or Coleoptera orders, and is sufficiently homologous tothe protein of SEQ ID NO: 2. A variety of PIP-45-2 polypeptides arecontemplated. One source of a PIP-45-2 polypeptide or related proteinsis a bacterial strain that contains the polynucleotide of SEQ ID NO:109, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO:143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 221 orSEQ ID NO: 223 that encode the PIP-45-2 polypeptide of SEQ ID NO: 2, SEQID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28,SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO:40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 235 and SEQ ID NO: 237,respectively. One source of a PIP-45-2 polypeptide or related proteinsis from a Pseudomonas, Thalassuspira, Paracoccusor Cellvibrio strain.One source of a PIP-45-2 polypeptide or related proteins is from aPseudomonas strain selected from but not limited to Pseudomonasbrenneri, Pseudomonas monteilii, Pseudomonas gessardii, Pseudomonasplecoglossicida, Pseudomonas putida, Pseudomonas poae,Pseudomonastrivialis, Pseudomonaslibanensis, Pseudomonas fluorescens andPseudomonas asplenii.

In some embodiments a PIP-45-2 polypeptide is sufficiently homologous tothe amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 20,SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ IDNO: 46, SEQ ID NO: 235 or SEQ ID NO: 237 and has insecticidal activity.“Sufficiently homologous” is used herein to refer to an amino acidsequence that has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greatersequence homology compared to a reference sequence using one of thealignment programs described herein using standard parameters. One ofskill in the art will recognize that these values can be appropriatelyadjusted to determine corresponding homology of proteins taking intoaccount amino acid similarity and the like. In some embodiments thesequence homology is against the full length sequence of a PIP-45-2polypeptide. In some embodiments the PIP-45-2 polypeptide has at leastabout 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity comparedto SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ IDNO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 235 orSEQ ID NO: 237.

In some embodiments the PIP-45-2 polypeptide has at least 99.2% orgreater sequence identity compared to SEQ ID NO: 2. In some embodimentsthe PIP-45-2 polypeptide has at least 98.5% or greater sequence identitycompared to SEQ ID NO: 18. In some embodiments the PIP-45-2 polypeptidehas at least 96% or greater sequence identity compared to SEQ ID NO: 20.In some embodiments the PIP-45-2 polypeptide has at least 80% or greatersequence identity compared to SEQ ID NO: 22. In some embodiments thePIP-45-2 polypeptide has at least 81% or greater sequence identitycompared to SEQ ID NO: 24. In some embodiments the PIP-45-2 polypeptidehas at least 99.5% or greater sequence identity compared to SEQ ID NO:28. In some embodiments the PIP-45-2 polypeptide has at least 98.5% orgreater sequence identity compared to SEQ ID NO: 30. In some embodimentsthe PIP-45-2 polypeptide has at least 92% or greater sequence identitycompared to SEQ ID NO: 32. In some embodiments the PIP-45-2 polypeptidehas at least 91.5% or greater sequence identity compared to SEQ ID NO:34. In some embodiments the PIP-45-2 polypeptide has at least 70% orgreater sequence identity compared to SEQ ID NO: 36. In some embodimentsthe PIP-45-2 polypeptide has at least 90% or greater sequence identitycompared to SEQ ID NO: 40. In some embodiments the PIP-45-2 polypeptidehas at least 94% or greater sequence identity compared to SEQ ID NO: 44.In some embodiments the PIP-45-2 polypeptide has at least 70% or greatersequence identity compared to SEQ ID NO: 46.

PIP-64-1 polypeptides are encompassed by the disclosure. “PseudomonasInsecticidal Protein-64-1”, “PIP-64-1 polypeptide” or “PIP-64-1 protein”as used herein interchangeably refers to a polypeptide havinginsecticidal activity against one or more insect pests of theLepidoptera and/or Coleoptera orders, and is sufficiently homologous tothe protein of SEQ ID NO: 53. A variety of PIP-64-1 polypeptides arecontemplated. One source of a PIP-64-1 polypeptide or related proteinsis a bacterial strain that contains the polynucleotide of SEQ ID NO:160, SEQ ID NO: 165 or SEQ ID NO: 224 that encode the PIP-64-1polypeptide of SEQ ID NO: 53, SEQ ID NO: 58 and SEQ ID NO: 238. Onesource of a PIP-64-1 polypeptide or related proteins is from aPseudomonas, Enterobacter or Alcaligenes strain. One source of aPIP-64-1 polypeptide or related proteins is from a Pseudomonas orAlcaligenes strain selected from but not limited to Pseudomonasbrenneri, Pseudomonas gessardii, Pseudomonas fluorescens, Pseudomonasbrassicacearum, Pseudomonas entomophila and Alcaligenes faecalis.

In some embodiments a PIP-64-1 polypeptide is sufficiently homologous tothe amino acid sequence of SEQ ID NO: 53, SEQ ID NO: 58 or SEQ ID NO:238 and has insecticidal activity. “Sufficiently homologous” is usedherein to refer to an amino acid sequence that has at least about 50%,55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater sequence homology compared to areference sequence using one of the alignment programs described hereinusing standard parameters. One of skill in the art will recognize thatthese values can be appropriately adjusted to determine correspondinghomology of proteins taking into account amino acid similarity and thelike. In some embodiments the sequence homology is against the fulllength sequence of a PIP-64-1 polypeptide. In some embodiments thePIP-64-1 polypeptide has at least about 50%, 55%, 60%, 65%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to SEQ ID NO: 53, SEQ ID NO: 58 orSEQ ID NO: 238.

In some embodiments the PIP-64-1 polypeptide has at least 75% or greatersequence identity compared to SEQ ID NO: 53. In some embodiments thePIP-64-1 polypeptide has at least 99.7% or greater sequence identitycompared to SEQ ID NO: 58. In some embodiments the PIP-64-1 polypeptidehas at least 75% or greater sequence identity compared to SEQ ID NO:238.

PIP-64-2 polypeptides are encompassed by the disclosure. “PseudomonasInsecticidal Protein-64-2”, “PIP-64-2 polypeptide” or “PIP-64-2 protein”as used herein interchangeably refers to a polypeptide havinginsecticidal activity against one or more insect pests of theLepidoptera and/or Coleoptera orders, and is sufficiently homologous tothe protein of SEQ ID NO:54. A variety of PIP-64-2 polypeptides arecontemplated. One source of a PIP-64-2 polypeptide or related proteinsis a bacterial strain that contains the polynucleotide of SEQ ID NO:161, SEQ ID NO: 162, SEQ ID NO: 166 or SEQ ID NO: 225 that encode thePIP-64-2 polypeptide of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 59 andSEQ ID NO: 239, respectively. One source of a PIP-64-2 polypeptide orrelated proteins is from a Pseudomonas, Enterobacter or Alcaligenesstrain. One source of a PIP-64-2 polypeptide or related proteins is froma Pseudomonas or Alcaligenes strain selected from but not limited toPseudomonas brenneri, Pseudomonas gessardii, Pseudomonas fluorescens,Pseudomonas brassicacearum, Pseudomonas entomophila and Alcaligenesfaecalis.

In some embodiments a PIP-64-2 polypeptide is sufficiently homologous tothe amino acid sequence of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 59or SEQ ID NO: 239 and has insecticidal activity. “Sufficientlyhomologous” is used herein to refer to an amino acid sequence that hasat least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homologycompared to a reference sequence using one of the alignment programsdescribed herein using standard parameters. One of skill in the art willrecognize that these values can be appropriately adjusted to determinecorresponding homology of proteins taking into account amino acidsimilarity and the like. In some embodiments the sequence homology isagainst the full length sequence of a PIP-64-2 polypeptide. In someembodiments the PIP-64-2 polypeptide has at least about 50%, 55%, 60%,65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or greater sequence identity compared to SEQ ID NO: 54,SEQ ID NO: 55, SEQ ID NO: 59 or SEQ ID NO: 239.

In some embodiments the PIP-64-2 polypeptide has at least 70% or greatersequence identity compared to SEQ ID NO: 54. In some embodiments thePIP-64-2 polypeptide has at least 70% or greater sequence identitycompared to SEQ ID NO: 55. In some embodiments the PIP-64-2 polypeptidehas at least 91% or greater sequence identity compared to SEQ ID NO: 59.In some embodiments the PIP-64-2 polypeptide has at least 70% or greatersequence identity compared to SEQ ID NO: 239.

PIP-74-1 polypeptides are encompassed by the disclosure. “PseudomonasInsecticidal Protein-74-1”, “PIP-74-1 polypeptide” or “PIP-74-1 protein”as used herein interchangeably refers to a polypeptide havinginsecticidal activity against one or more insect pests of theLepidoptera and/or Coleoptera orders, and is sufficiently homologous tothe protein of SEQ ID NO: 73. A variety of PIP-74-1 polypeptides arecontemplated. One source of a PIP-74-1 polypeptide or related proteinsis a bacterial strain that contains the polynucleotide of SEQ ID NO:180, SEQ ID NO: 182 or SEQ ID NO: 184 that encode the PIP-74-1polypeptide of SEQ ID NO: 73, SEQ ID NO: 75 and SEQ ID NO: 77,respectively. One source of a PIP-74-1 polypeptide or related proteinsis from a Pseudomonas strain. One source of a PIP-74-1 polypeptide orrelated proteins is from a Pseudomonas strain selected from but notlimited to Pseudomonas rhodesiae and Pseudomonas orientalis.

In some embodiments a PIP-74-1 polypeptide is sufficiently homologous tothe amino acid sequence of SEQ ID NO: 73, SEQ ID NO: 75 or SEQ ID NO: 77and has insecticidal activity. “Sufficiently homologous” is used hereinto refer to an amino acid sequence that has at least about 50%, 55%,60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater sequence homology compared to a referencesequence using one of the alignment programs described herein usingstandard parameters. One of skill in the art will recognize that thesevalues can be appropriately adjusted to determine corresponding homologyof proteins taking into account amino acid similarity and the like. Insome embodiments the sequence homology is against the full lengthsequence of a PIP-74-1 polypeptide. In some embodiments the PIP-74-1polypeptide has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greatersequence identity compared to SEQ ID NO: 73, SEQ ID NO: 75 or SEQ ID NO:77.

In some embodiments the PIP-74-1 polypeptide has at least 75% or greatersequence identity compared to SEQ ID NO: 73. In some embodiments thePIP-74-1 polypeptide has at least 75% or greater sequence identitycompared to SEQ ID NO: 75. In some embodiments the PIP-74-1 polypeptidehas at least 75% or greater sequence identity compared to SEQ ID NO: 77.

PIP-74-2 polypeptides are encompassed by the disclosure. “PseudomonasInsecticidal Protein-74-2”, “PIP-74-2 polypeptide” or “PIP-74-2 protein”as used herein interchangeably refers to a polypeptide havinginsecticidal activity against one or more insect pests of theLepidoptera and/or Coleoptera orders, and is sufficiently homologous tothe protein of SEQ ID NO: 74. A variety of PIP-74-2 polypeptides arecontemplated. One source of a PIP-74-2 polypeptide or related proteinsis a bacterial strain that contains the polynucleotide of SEQ ID NO:181, SEQ ID NO: 183, SEQ ID NO: 185 that encode the PIP-74-2 polypeptideof SEQ ID NO: 74, SEQ ID NO: 76 and SEQ ID NO: 78, respectively. Onesource of a PIP-74-2 polypeptide or related proteins is from aPseudomonas strain. One source of a PIP-74-2 polypeptide or relatedproteins is from a Pseudomonas strain selected from but not limited toPseudomonas rhodesiae and Pseudomonas orientalis.

In some embodiments a PIP-74-2 polypeptide is sufficiently homologous tothe amino acid sequence of SEQ ID NO: 74, SEQ ID NO: 76 or SEQ ID NO: 78and has insecticidal activity. “Sufficiently homologous” is used hereinto refer to an amino acid sequence that has at least about 50%, 55%,60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater sequence homology compared to a referencesequence using one of the alignment programs described herein usingstandard parameters. One of skill in the art will recognize that thesevalues can be appropriately adjusted to determine corresponding homologyof proteins taking into account amino acid similarity and the like. Insome embodiments the sequence homology is against the full lengthsequence of a PIP-74-2 polypeptide. In some embodiments the PIP-74-2polypeptide has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greatersequence identity compared to SEQ ID NO: 74, SEQ ID NO: 76 or SEQ ID NO:78.

In some embodiments the PIP-74-2 polypeptide has at least 75% or greatersequence identity compared to SEQ ID NO: 74. In some embodiments thePIP-74-2 polypeptide has at least 75% or greater sequence identitycompared to SEQ ID NO: 76. In some embodiments the PIP-74-2 polypeptidehas at least 75% or greater sequence identity compared to SEQ ID NO: 78.

PIP-75 polypeptides are encompassed by the disclosure. “PseudomonasInsecticidal Protein-75”, “PIP-75 polypeptide” or “PIP-75 protein” asused herein interchangeably refers to a polypeptide having insecticidalactivity against one or more insect pests of the Lepidoptera and/orColeoptera orders, and is sufficiently homologous to the protein of SEQID NO: 79. A variety of PIP-75 polypeptides are contemplated. One sourceof a PIP-75 polypeptide or related proteins is a bacterial strain thatcontains the polynucleotide of SEQ ID NO: 186, SEQ ID NO: 187, SEQ IDNO: 188, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193 or SEQ ID NO:194 that encode the PIP-75 polypeptide of SEQ ID NO: 79, SEQ ID NO: 80,SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 and SEQ IDNO: 87, respectively. One source of a PIP-75 polypeptide or relatedproteins is from a Pseudomonas, Enterobacter or Serratia strain. Onesource of a PIP-75 polypeptide or related proteins is from aPseudomonas, Enterobacter or Serratia strain selected from but notlimited to Pseudomonas antarctica, Pseudomonas orientalis, Enterobacterasburiae, Serratia plymuthica, and Serratia liquefaciens.

In some embodiments a PIP-75 polypeptide is sufficiently homologous tothe amino acid sequence of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81,SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87 and hasinsecticidal activity. “Sufficiently homologous” is used herein to referto an amino acid sequence that has at least about 50%, 55%, 60%, 65%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater sequence homology compared to a reference sequenceusing one of the alignment programs described herein using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding homology ofproteins taking into account amino acid similarity and the like. In someembodiments the sequence homology is against the full length sequence ofa PIP-75 polypeptide. In some embodiments the PIP-75 polypeptide has atleast about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identitycompared to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84,SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87.

In some embodiments the PIP-75 polypeptide has at least 75% or greatersequence identity compared to SEQ ID NO: 79. In some embodiments thePIP-75 polypeptide has at least 75% or greater sequence identitycompared to SEQ ID NO: 80. In some embodiments the PIP-75 polypeptidehas at least 86% or greater sequence identity compared to SEQ ID NO: 81.In some embodiments the PIP-75 polypeptide has at least 75% or greatersequence identity compared to SEQ ID NO: 84. In some embodiments thePIP-75 polypeptide has at least 75% or greater sequence identitycompared to SEQ ID NO: 85. In some embodiments the PIP-75 polypeptidehas at least 75% or greater sequence identity compared to SEQ ID NO: 86.In some embodiments the PIP-75 polypeptide has at least 75% or greatersequence identity compared to SEQ ID NO: 87.

PIP-77 polypeptides are encompassed by the disclosure. “PseudomonasInsecticidal Protein-77”, “PIP-77 polypeptide” or “PIP-77 protein” asused herein interchangeably refers to a polypeptide having insecticidalactivity against one or more insect pests of the Lepidoptera and/orColeoptera orders, and is sufficiently homologous to the protein of SEQID NO: 88. A variety of PIP-77 polypeptides are contemplated. One sourceof a PIP-77 polypeptide or related proteins is a bacterial strain thatcontains the polynucleotide of SEQ ID NO: 195, SEQ ID NO:196, SEQ IDNO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO: 200, SEQ ID NO: 201,SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ IDNO: 207, SEQ ID NO: 227, SEQ ID NO: 228 or SEQ ID NO: 231 that encodethe PIP-77 polypeptide of SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90,SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO:96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 241, SEQ IDNO: 242 and SEQ ID NO: 245, respectively. One source of a PIP-77polypeptide or related proteins is from a Pseudomonas, Enterobacter,Shewanella, Haemophilus or Aeromonas strain. One source of a PIP-77polypeptide or related proteins is from a Pseudomonas strain selectedfrom but not limited to Pseudomonas chlororaphis, Pseudomonasbrassicacearum, Pseudomonas fluorescens and Pseudomonas rhodesiae.

In some embodiments a PIP-77 polypeptide is sufficiently homologous tothe amino acid sequence of SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90,SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO:96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 241, SEQ IDNO: 242 or SEQ ID NO: 245 and has insecticidal activity. “Sufficientlyhomologous” is used herein to refer to an amino acid sequence that hasat least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homologycompared to a reference sequence using one of the alignment programsdescribed herein using standard parameters. One of skill in the art willrecognize that these values can be appropriately adjusted to determinecorresponding homology of proteins taking into account amino acidsimilarity and the like. In some embodiments the sequence homology isagainst the full length sequence of a PIP-77 polypeptide. In someembodiments the PIP-77 polypeptide has at least about 50%, 55%, 60%,65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or greater sequence identity compared to SEQ ID NO: 88,SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO:94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ IDNO: 100, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 245.

In some embodiments the PIP-77 polypeptide has at least 93% or greatersequence identity compared to SEQ ID NO: 88. In some embodiments thePIP-77 polypeptide has at least 97% or greater sequence identitycompared to SEQ ID NO: 89. In some embodiments the PIP-77 polypeptidehas at least 99% or greater sequence identity compared to SEQ ID NO: 90.In some embodiments the PIP-77 polypeptide has at least 97% or greatersequence identity compared to SEQ ID NO: 92. In some embodiments thePIP-77 polypeptide has at least 87% or greater sequence identitycompared to SEQ ID NO: 93. In some embodiments the PIP-77 polypeptidehas at least 86% or greater sequence identity compared to SEQ ID NO: 94.In some embodiments the PIP-77 polypeptide has at least 85% or greatersequence identity compared to SEQ ID NO: 95. In some embodiments thePIP-77 polypeptide has at least 84% or greater sequence identitycompared to SEQ ID NO: 96. In some embodiments the PIP-77 polypeptidehas at least 85% or greater sequence identity compared to SEQ ID NO: 97.In some embodiments the PIP-77 polypeptide has at least 83% or greatersequence identity compared to SEQ ID NO: 98. In some embodiments thePIP-77 polypeptide has at least 80% or greater sequence identitycompared to SEQ ID NO: 100. In some embodiments the PIP-77 polypeptidehas at least 85% or greater sequence identity compared to SEQ ID NO:241. In some embodiments the PIP-77 polypeptide has at least 83% orgreater sequence identity compared to SEQ ID NO: 242. In someembodiments the PIP-77 polypeptide has at least 96% or greater sequenceidentity compared to SEQ ID NO: 245.

As used herein, the terms “protein,” “peptide molecule,” or“polypeptide” includes any molecule that comprises five or more aminoacids. It is well known in the art that protein, peptide or polypeptidemolecules may undergo modification, including post-translationalmodifications, such as, but not limited to, disulfide bond formation,glycosylation, phosphorylation or oligomerization. Thus, as used herein,the terms “protein,” “peptide molecule” or “polypeptide” includes anyprotein that is modified by any biological or non-biological process.The terms “amino acid” and “amino acids” refer to all naturallyoccurring L-amino acids.

In some embodiments a PIP-45-1 polypeptide has a calculated molecularweight of between about 40 kDa and about 80 kDa, between about 50 kDaand about 70 kDa, between about 60 kDa and about 65 kDa, between about61 kDa and about 64 kDa, beteewn about 62 kDa and about 63 kDa, andbetween about 62.25 kDa, about 62.75 kDa. As used herein, the term“about” used in the context of molecular weight of an insecticidalpolypeptide means±0.25 kilodaltons.

In some embodiments a PIP-45-2 polypeptide has a calculated molecularweight of between about 40 kDa and about 80 kDa, between about 50 kDaand about 64 kDa, between about 55 kDa and about 60 kDa, between about56.5 kDa and about 59 kDa, and between about 57.25 kDa and about 58 kDa.

In some embodiments a PIP-64-1 polypeptide has a calculated molecularweight of between about 20 kDa and about 40 kDa, between about 25 kDaand about 32 kDa, between about 26 kDa and about 31 kDa, between about27 kDa and about 30 kDa, between about 28 kDa and about 29 kDa, andbetween about 28.1 kDa and about 28.7 kDa.

In some embodiments a PIP-64-2 polypeptide has a calculated molecularweight of between about 20 kDa and about 40 kDa, between about 25 kDaand about 32 kDa, between about 26 kDa and about 31 kDa, between about27 kDa and about 30 kDa, and between about 28.25 kDa and about 29 kDa.

In some embodiments a PIP-74-1 polypeptide has a calculated molecularweight of between about 40 kDa and about 80 kDa, between about 50 kDaand about 70 kDa, beteewn about 55 kDa and about 73 kDa, beteewn about57 kDa and about 61 kDa, beteewn about 58 kDa and about 60 kDa andbetween about 58.75 kDa, about 59.25 kDa. As used herein, the term“about” used in the context of molecular weight of an insecticidalpolypeptide means±0.25 kilodaltons.

In some embodiments a PIP-74-2 polypeptide has a calculated molecularweight of between about 35 kDa and about 65 kDa, between about 45 kDaand about 51.5 kDa, between about 47.5 kDa and about 49.5 kDa, andbetween about 48.25 kDa and about 48.75 kDa.

In some embodiments a PIP-75 polypeptide has a calculated molecularweight of between about 6 kDa and about 14 kDa, between about 8 kDa andabout 13.5 kDa, between about 9 kDa and about 12 kDa, between about 9.5kDa and about 11.5 kDa, and between about 10.4 kDa and about 10.8 kDa.

In some embodiments a PIP-77 polypeptide has a calculated molecularweight of between about 7 kDa and about 13 kDa, between about 8 kDa andabout 12 kDa, between about 9 kDa and about 11 kDa, between about 9.5kDa and about 10.3 kDa, and between about 9.75 kDa and about 10.25 kDa.

In some embodiments the insecticidal polypeptides of the disclosure havea modified physical property. As used herein, the term “physicalproperty” refers to any parameter suitable for describing thephysical-chemical characteristics of a protein. As used herein,“physical property of interest” and “property of interest” are usedinterchangeably to refer to physical properties of proteins that arebeing investigated and/or modified. Examples of physical propertiesinclude, but are not limited to net surface charge and chargedistribution on the protein surface, net hydrophobicity and hydrophobicresidue distribution on the protein surface, surface charge density,surface hydrophobicity density, total count of surface ionizable groups,surface tension, protein size and its distribution in solution, meltingtemperature, heat capacity, and second virial coefficient. Examples ofphysical properties also include, but are not limited to solubility,folding, stability, and digestibility. In some embodiments theinsecticidal polypeptides of the dislcosure have increased digestibilityof proteolytic fragments in an insect gut. Models for digestion bysimulated simulated gastric fluids are known to one skilled in the art(Fuchs, R. L. and J. D. Astwood. Food Technology 50: 83-88, 1996;Astwood, J. D., et al Nature Biotechnology 14: 1269-1273, 1996; Fu T Jet al J. Agric Food Chem. 50: 7154-7160, 2002).

In some embodiments variants include polypeptides that differ in aminoacid sequence due to mutagenesis. Variant proteins encompassed by thedisclosure are biologically active, that is they continue to possess thedesired biological activity (i.e. pesticidal activity) of the nativeprotein. In some embodiment the variant will have at least about 10%, atleast about 30%, at least about 50%, at least about 70%, at least about80% or more of the insecticidal activity of the native protein. In someembodiments, the variants may have improved activity over the nativeprotein.

Bacterial genes quite often possess multiple methionine initiationcodons in proximity to the start of the open reading frame. Often,translation initiation at one or more of these start codons will lead togeneration of a functional protein. These start codons can include ATGcodons. However, bacteria such as Bacillus sp. also recognize the codonGTG as a start codon, and proteins that initiate translation at GTGcodons contain a methionine at the first amino acid. On rare occasions,translation in bacterial systems can initiate at a TTG codon, though inthis event the TTG encodes a methionine. Furthermore, it is not oftendetermined a priori which of these codons are used naturally in thebacterium. Thus, it is understood that use of one of the alternatemethionine codons may also lead to generation of pesticidal proteins.These pesticidal proteins are encompassed in the present disclosure andmay be used in the methods of the present disclosure. It will beunderstood that, when expressed in plants, it will be necessary to alterthe alternate start codon to ATG for proper translation.

In another aspect the insecticidal polypeptide of the disclsosure may beexpressed as a precursor protein with an intervening sequence thatcatalyzes multi-step, post translational protein splicing. Proteinsplicing involves the excision of an intervening sequence from apolypeptide with the concomitant joining of the flanking sequences toyield a new polypeptide (Chong, et al., (1996) J. Biol. Chem.,271:22159-22168). This intervening sequence or protein splicing element,referred to as inteins, which catalyze their own excision through threecoordinated reactions at the N-terminal and C-terminal splice junctions:an acyl rearrangement of the N-terminal cysteine or serine; atransesterfication reaction between the two termini to form a branchedester or thioester intermediate and peptide bond cleavage coupled tocyclization of the intein C-terminal asparagine to free the intein(Evans, et al., (2000) J. Biol. Chem., 275:9091-9094. The elucidation ofthe mechanism of protein splicing has led to a number of intein-basedapplications (Comb, et al., U.S. Pat. No. 5,496,714; Comb, et al., U.S.Pat. No. 5,834,247; Camarero and Muir, (1999) J. Amer. Chem. Soc.121:5597-5598; Chong, et al., (1997) Gene 192:271-281, Chong, et al.,(1998) Nucleic Acids Res. 26:5109-5115; Chong, et al., (1998) J. Biol.Chem. 273:10567-10577; Cotton, et al., (1999) J. Am. Chem. Soc.121:1100-1101; Evans, et al., (1999) J. Biol. Chem. 274:18359-18363;Evans, et al., (1999) J. Biol. Chem. 274:3923-3926; Evans, et al.,(1998) Protein Sci. 7:2256-2264; Evans, et al., (2000) J. Biol. Chem.275:9091-9094; Iwai and Pluckthun, (1999) FEBS Lett. 459:166-172;Mathys, et al., (1999) Gene 231:1-13; Mills, et al., (1998) Proc. Natl.Acad. Sci. USA 95:3543-3548; Muir, et al., (1998) Proc. Natl. Acad. Sci.USA 95:6705-6710; Otomo, et al., (1999) Biochemistry 38:16040-16044;Otomo, et al., (1999) J. Biolmol. NMR 14:105-114; Scott, et al., (1999)Proc. Natl. Acad. Sci. USA 96:13638-13643; Severinov and Muir, (1998) J.Biol. Chem. 273:16205-16209; Shingledecker, et al., (1998) Gene207:187-195; Southworth, et al., (1998) EMBO J. 17:918-926; Southworth,et al., (1999) Biotechniques 27:110-120; Wood, et al., (1999) Nat.Biotechnol. 17:889-892; Wu, et al., (1998a) Proc. Natl. Acad. Sci. USA95:9226-9231; Wu, et al., (1998b) Biochim Biophys Acta 1387:422-432; Xu,et al., (1999) Proc. Natl. Acad. Sci. USA 96:388-393; Yamazaki, et al.,(1998) J. Am. Chem. Soc., 120:5591-5592). For the application of inteinsin plant transgenes, see, Yang, et al., (Transgene Res 15:583-593(2006)) and Evans, et al., (Annu. Rev. Plant Biol. 56:375-392 (2005)).

In another aspect the insecticidal polypeptide of the disclosure may beencoded by two separate genes where the intein of the precursor proteincomes from the two genes, referred to as a split-intein, and the twoportions of the precursor are joined by a peptide bond formation. Thispeptide bond formation is accomplished by intein-mediatedtrans-splicing. For this purpose, a first and a second expressioncassette comprising the two separate genes further code for inteinscapable of mediating protein trans-splicing. By trans-splicing, theproteins and polypeptides encoded by the first and second fragments maybe linked by peptide bond formation. Trans-splicing inteins may beselected from the nucleolar and organellar genomes of differentorganisms including eukaryotes, archaebacteria and eubacteria. Inteinsthat may be used for are listed at neb.com/neb/inteins.html, which canbe accessed on the world-wide web using the “www” prefix). Thenucleotide sequence coding for an intein may be split into a 5′ and a 3′part that code for the 5′ and the 3′ part of the intein, respectively.Sequence portions not necessary for intein splicing (e.g. homingendonuclease domain) may be deleted. The intein coding sequence is splitsuch that the 5′ and the 3′ parts are capable of trans-splicing. Forselecting a suitable splitting site of the intein coding sequence, theconsiderations published by Southworth, et al., (1998) EMBO J.17:918-926 may be followed. In constructing the first and the secondexpression cassette, the 5′ intein coding sequence is linked to the 3′end of the first fragment coding for the N-terminal part of theinsecticidal polypeptide of the disclosure and the 3′ intein codingsequence is linked to the 5′ end of the second fragment coding for theC-terminal part of the insecticidal polypeptide of the disclosure.

In general, the trans-splicing partners can be designed using any splitintein, including any naturally-occurring or artificially-split splitintein. Several naturally-occurring split inteins are known, forexample: the split intein of the DnaE gene of Synechocystis sp. PCC6803(see, Wu, et al., (1998) Proc Natl Acad Sci USA. 95(16):9226-31 andEvans, et al., (2000) J Biol Chem. 275(13):9091-4 and of the DnaE genefrom Nostoc punctiforme (see, Iwai, et al., (2006) FEBS Lett.580(7):1853-8). Non-split inteins have been artificially split in thelaboratory to create new split inteins, for example: the artificiallysplit Ssp DnaB intein (see, Wu, et al., (1998) Biochim Biophys Acta.1387:422-32) and split Sce VMA intein (see, Brenzel, et al., (2006)Biochemistry. 45(6):1571-8) and an artificially split fungal mini-intein(see, Elleuche, et al., (2007) Biochem Biophys Res Commun.355(3):830-4). There are also intein databases available that catalogueknown inteins (see for example the online-database available at:bioinformatics.weizmann.ac.ilrpietro/inteins/Inteinstable.html, whichcan be accessed on the world-wide web using the “www” prefix).

Naturally-occurring non-split inteins may have endonuclease or otherenzymatic activities that can typically be removed when designing anartificially-split split intein. Such mini-inteins or minimized splitinteins are well known in the art and are typically less than 200 aminoacid residues long (see, Wu, et al., (1998) Biochim Biophys Acta.1387:422-32). Suitable split inteins may have other purificationenabling polypeptide elements added to their structure, provided thatsuch elements do not inhibit the splicing of the split intein or areadded in a manner that allows them to be removed prior to splicing.Protein splicing has been reported using proteins that comprisebacterial intein-like (BIL) domains (see, Amitai, et al., (2003) MolMicrobiol. 47:61-73) and hedgehog (Hog) auto-processing domains (thelatter is combined with inteins when referred to as the Hog/inteinsuperfamily or HINT family (see, Dassa, et al., (2004) J Biol Chem.279:32001-7) and domains such as these may also be used to prepareartificially-split inteins. In particular, non-splicing members of suchfamilies may be modified by molecular biology methodologies to introduceor restore splicing activity in such related species. Recent studiesdemonstrate that splicing can be observed when a N-terminal split inteincomponent is allowed to react with a C-terminal split intein componentnot found in nature to be its “partner”; for example, splicing has beenobserved utilizing partners that have as little as 30 to 50% homologywith the “natural” splicing partner (see, Dassa, et al., (2007)Biochemistry. 46(1):322-30). Other such mixtures of disparate splitintein partners have been shown to be unreactive one with another (see,Brenzel, et al., (2006) Biochemistry. 45(6):1571-8). However, it iswithin the ability of a person skilled in the relevant art to determinewhether a particular pair of polypeptides is able to associate with eachother to provide a functional intein, using routine methods and withoutthe exercise of inventive skill.

In another aspect the insecticidal polypeptide of the disclosure is acircular permuted variant. The development of recombinant DNA methodshas made it possible to study the effects of sequence transposition onprotein folding, structure and function. The approach used in creatingnew sequences resembles that of naturally occurring pairs of proteinsthat are related by linear reorganization of their amino acid sequences(Cunningham, et al., (1979) Proc. Natl. Acad. Sci. U.S.A. 76:3218-3222;Teather and Erfle, (1990) J. Bacteriol. 172:3837-3841; Schimming, etal., (1992) Eur. J. Biochem. 204:13-19; Yamiuchi and Minamikawa, (1991)FEBS Lett. 260:127-130; MacGregor, et al., (1996) FEBS Lett.378:263-266). The first in vitro application of this type ofrearrangement to proteins was described by Goldenberg and Creighton (J.Mol. Biol. 165:407-413, 1983). In creating a circular permuted variant anew N-terminus is selected at an internal site (breakpoint) of theoriginal sequence, the new sequence having the same order of amino acidsas the original from the breakpoint until it reaches an amino acid thatis at or near the original C-terminus. At this point the new sequence isjoined, either directly or through an additional portion of sequence(linker), to an amino acid that is at or near the original N-terminusand the new sequence continues with the same sequence as the originaluntil it reaches a point that is at or near the amino acid that wasN-terminal to the breakpoint site of the original sequence, this residueforming the new C-terminus of the chain. The length of the amino acidsequence of the linker can be selected empirically or with guidance fromstructural information or by using a combination of the two approaches.When no structural information is available, a small series of linkerscan be prepared for testing using a design whose length is varied inorder to span a range from 0 to 50 Å and whose sequence is chosen inorder to be consistent with surface exposure (hydrophilicity, Hopp andWoods, (1983) Mol. lmmunol. 20:483-489; Kyte and Doolittle, (1982) J.Mol. Biol. 157:105-132; solvent exposed surface area, Lee and Richards,(1971) J. Mol. Biol. 55:379-400) and the ability to adopt the necessaryconformation without deranging the configuration of the pesticidalpolypeptide (conformationally flexible; Karplus and Schulz, (1985)Naturwissenschaften 72:212-213). Assuming an average of translation of2.0 to 3.8 Å per residue, this would mean the length to test would bebetween 0 to 30 residues, with 0 to 15 residues being the preferredrange. Exemplary of such an empirical series would be to constructlinkers using a cassette sequence such as Gly-Gly-Gly-Ser repeated ntimes, where n is 1, 2, 3 or 4. Those skilled in the art will recognizethat there are many such sequences that vary in length or compositionthat can serve as linkers with the primary consideration being that theybe neither excessively long nor short (cf., Sandhu, (1992) Critical Rev.Biotech. 12:437-462); if they are too long, entropy effects will likelydestabilize the three-dimensional fold, and may also make foldingkinetically impractical, and if they are too short, they will likelydestabilize the molecule because of torsional or steric strain. Thoseskilled in the analysis of protein structural information will recognizethat using the distance between the chain ends, defined as the distancebetween the c-alpha carbons, can be used to define the length of thesequence to be used or at least to limit the number of possibilitiesthat must be tested in an empirical selection of linkers. They will alsorecognize that it is sometimes the case that the positions of the endsof the polypeptide chain are ill-defined in structural models derivedfrom x-ray diffraction or nuclear magnetic resonance spectroscopy data,and that when true, this situation will therefore need to be taken intoaccount in order to properly estimate the length of the linker required.From those residues whose positions are well defined are selected tworesidues that are close in sequence to the chain ends, and the distancebetween their c-alpha carbons is used to calculate an approximate lengthfor a linker between them. Using the calculated length as a guide,linkers with a range of number of residues (calculated using 2 to 3.8 Åper residue) are then selected. These linkers may be composed of theoriginal sequence, shortened or lengthened as necessary, and whenlengthened the additional residues may be chosen to be flexible andhydrophilic as described above; or optionally the original sequence maybe substituted for using a series of linkers, one example being theGly-Gly-Gly-Ser cassette approach mentioned above; or optionally acombination of the original sequence and new sequence having theappropriate total length may be used. Sequences of pesticidalpolypeptides capable of folding to biologically active states can beprepared by appropriate selection of the beginning (amino terminus) andending (carboxyl terminus) positions from within the originalpolypeptide chain while using the linker sequence as described above.Amino and carboxyl termini are selected from within a common stretch ofsequence, referred to as a breakpoint region, using the guidelinesdescribed below. A novel amino acid sequence is thus generated byselecting amino and carboxyl termini from within the same breakpointregion. In many cases the selection of the new termini will be such thatthe original position of the carboxyl terminus immediately preceded thatof the amino terminus. However, those skilled in the art will recognizethat selections of termini anywhere within the region may function, andthat these will effectively lead to either deletions or additions to theamino or carboxyl portions of the new sequence. It is a central tenet ofmolecular biology that the primary amino acid sequence of a proteindictates folding to the three-dimensional structure necessary forexpression of its biological function. Methods are known to thoseskilled in the art to obtain and interpret three-dimensional structuralinformation using x-ray diffraction of single protein Crystals ornuclear magnetic resonance spectroscopy of protein solutions. Examplesof structural information that are relevant to the identification ofbreakpoint regions include the location and type of protein secondarystructure (alpha and 3-10 helices, parallel and anti-parallel betasheets, chain reversals and turns, and loops; Kabsch and Sander, (1983)Biopolymers 22:2577-2637; the degree of solvent exposure of amino acidresidues, the extent and type of interactions of residues with oneanother (Chothia, (1984) Ann. Rev. Biochem. 53:537-572) and the staticand dynamic distribution of conformations along the polypeptide chain(Alber and Mathews, (1987) Methods Enzymol. 154:511-533). In some casesadditional information is known about solvent exposure of residues; oneexample is a site of post-translational attachment of carbohydrate whichis necessarily on the surface of the protein. When experimentalstructural information is not available or is not feasible to obtain,methods are also available to analyze the primary amino acid sequence inorder to make predictions of protein tertiary and secondary structure,solvent accessibility and the occurrence of turns and loops. Biochemicalmethods are also sometimes applicable for empirically determiningsurface exposure when direct structural methods are not feasible; forexample, using the identification of sites of chain scission followinglimited proteolysis in order to infer surface exposure (Gentile andSalvatore, (1993) Eur. J. Biochem. 218:603-621). Thus using either theexperimentally derived structural information or predictive methods(e.g., Srinivisan and Rose, (1995) Proteins: Struct., Funct. & Genetics22:81-99) the parental amino acid sequence is inspected to classifyregions according to whether or not they are integral to the maintenanceof secondary and tertiary structure. The occurrence of sequences withinregions that are known to be involved in periodic secondary structure(alpha and 3-10 helices, parallel and anti-parallel beta sheets) areregions that should be avoided. Similarly, regions of amino acidsequence that are observed or predicted to have a low degree of solventexposure are more likely to be part of the so-called hydrophobic core ofthe protein and should also be avoided for selection of amino andcarboxyl termini. In contrast, those regions that are known or predictedto be in surface turns or loops, and especially those regions that areknown not to be required for biological activity, are the preferredsites for location of the extremes of the polypeptide chain. Continuousstretches of amino acid sequence that are preferred based on the abovecriteria are referred to as a breakpoint region. Polynucleotidesencoding circular permuted insecticidal polypeptides of the disclosurewith new N-terminus/C-terminus which contain a linker region separatingthe original C-terminus and N-terminus can be made essentially followingthe method described in Mullins, et al., (1994) J. Am. Chem. Soc.116:5529-5533. Multiple steps of polymerase chain reaction (PCR)amplifications are used to rearrange the DNA sequence encoding theprimary amino acid sequence of the protein. Polynucleotides encodingcircular permuted insecticidal polypeptides of the disclosure with newN-terminus/C-terminus which contain a linker region separating theoriginal C-terminus and N-terminus can be made based on thetandem-duplication method described in Horlick, et al., (1992) ProteinEng. 5:427-431. Polymerase chain reaction (PCR) amplification of the newN-terminus/C-terminus genes is performed using a tandemly duplicatedtemplate DNA.

In another aspect fusion proteins are provided that include within itsamino acid sequence an amino acid sequence comprising an insecticidalpolypeptide of the disclosure. Methods for design and construction offusion proteins (and polynucleotides encoding same) are known to thoseof skill in the art. Polynucleotides encoding an insecticidalpolypeptide of the disclosure may be fused to signal sequences whichwill direct the localization of the insecticidal polypeptide of thedisclosure to insecticidal polypeptide of the embodiments from aprokaryotic or eukaryotic cell. For example, in E. coli, one may wish todirect the expression of the protein to the periplasmic space. Examplesof signal sequences or proteins (or fragments thereof) to which theinsecticidal polypeptide of the disclosure may be fused in order todirect the expression of the polypeptide to the periplasmic space ofbacteria include, but are not limited to, the pelB signal sequence, themaltose binding protein (MBP) signal sequence, MBP, the ompA signalsequence, the signal sequence of the periplasmic E. coli heat-labileenterotoxin B-subunit and the signal sequence of alkaline phosphatase.Several vectors are commercially available for the construction offusion proteins which will direct the localization of a protein, such asthe pMAL series of vectors (particularly the pMAL-p series) availablefrom New England Biolabs® (240 County Road, Ipswich, Mass. 01938-2723).In a specific embodiment, the insecticidal polypeptide of the disclosuremay be fused to the pelB pectate lyase signal sequence to increase theefficiency of expression and purification of such polypeptides inGram-negative bacteria (see, U.S. Pat. Nos. 5,576,195 and 5,846,818).Plant plastid transit peptide/polypeptide fusions are well known in theart (see, U.S. Pat. No. 7,193,133). Apoplast transit peptides such asrice or barley alpha-amylase secretion signal are also well known in theart. The plastid transit peptide is generally fused N-terminal to thepolypeptide to be targeted (e.g., the fusion partner). In oneembodiment, the fusion protein consists essentially of the plastidtransit peptide and the insecticidal polypeptide of the disclosure to betargeted. In another embodiment, the fusion protein comprises theplastid transit peptide and the polypeptide to be targeted. In suchembodiments, the plastid transit peptide is preferably at the N-terminusof the fusion protein. However, additional amino acid residues may beN-terminal to the plastid transit peptide providing that the fusionprotein is at least partially targeted to a plastid. In a specificembodiment, the plastid transit peptide is in the N-terminal half,N-terminal third or N-terminal quarter of the fusion protein. Most orall of the plastid transit peptide is generally cleaved from the fusionprotein upon insertion into the plastid. The position of cleavage mayvary slightly between plant species, at different plant developmentalstages, as a result of specific intercellular conditions or theparticular combination of transit peptide/fusion partner used. In oneembodiment, the plastid transit peptide cleavage is homogenous such thatthe cleavage site is identical in a population of fusion proteins. Inanother embodiment, the plastid transit peptide is not homogenous, suchthat the cleavage site varies by 1-10 amino acids in a population offusion proteins. The plastid transit peptide can be recombinantly fusedto a second protein in one of several ways. For example, a restrictionendonuclease recognition site can be introduced into the nucleotidesequence of the transit peptide at a position corresponding to itsC-terminal end and the same or a compatible site can be engineered intothe nucleotide sequence of the protein to be targeted at its N-terminalend. Care must be taken in designing these sites to ensure that thecoding sequences of the transit peptide and the second protein are kept“in frame” to allow the synthesis of the desired fusion protein. In somecases, it may be preferable to remove the initiator methionine codon ofthe second protein when the new restriction site is introduced. Theintroduction of restriction endonuclease recognition sites on bothparent molecules and their subsequent joining through recombinant DNAtechniques may result in the addition of one or more extra amino acidsbetween the transit peptide and the second protein. This generally doesnot affect targeting activity as long as the transit peptide cleavagesite remains accessible and the function of the second protein is notaltered by the addition of these extra amino acids at its N-terminus.Alternatively, one skilled in the art can create a precise cleavage sitebetween the transit peptide and the second protein (with or without itsinitiator methionine) using gene synthesis (Stemmer, et al., (1995) Gene164:49-53) or similar methods. In addition, the transit peptide fusioncan intentionally include amino acids downstream of the cleavage site.The amino acids at the N-terminus of the mature protein can affect theability of the transit peptide to target proteins to plastids and/or theefficiency of cleavage following protein import. This may be dependenton the protein to be targeted. See, e.g., Comai, et al., (1988) J. Biol.Chem. 263(29):15104-9.

In some embodiments fusion proteins are provide comprising aninsecticidal polypeptide of the disclosure, and an insecticidalpolypeptide joined by an amino acid linker.

In some embodiments fusion proteins are provided represented by aformula selected from the group consisting of:

R¹-L-R², R²-L-R¹, R¹-R² or R²-R¹

wherein R¹ is an insecticidal polypeptide of the disclosure. The R¹polypeptide is fused either directly or through a linker (L) segment tothe R² polypeptide. The term “directly” defines fusions in which thepolypeptides are joined without a peptide linker. Thus “L” represents achemical bound or polypeptide segment to which both R¹ and R² are fusedin frame, most commonly L is a linear peptide to which R¹ and R² arebound by amide bonds linking the carboxy terminus of R¹ to the aminoterminus of L and carboxy terminus of L to the amino terminus of R². By“fused in frame” is meant that there is no translation termination ordisruption between the reading frames of R¹ and R². The linking group(L) is generally a polypeptide of between 1 and 500 amino acids inlength. The linkers joining the two molecules are preferably designed to(1) allow the two molecules to fold and act independently of each other,(2) not have a propensity for developing an ordered secondary structurewhich could interfere with the functional domains of the two proteins,(3) have minimal hydrophobic or charged characteristic which couldinteract with the functional protein domains and (4) provide stericseparation of R¹ and R² such that R¹ and R² could interactsimultaneously with their corresponding receptors on a single cell.Typically surface amino acids in flexible protein regions include Gly,Asn and Ser. Virtually any permutation of amino acid sequencescontaining Gly, Asn and Ser would be expected to satisfy the abovecriteria for a linker sequence. Other neutral amino acids, such as Thrand Ala, may also be used in the linker sequence. Additional amino acidsmay also be included in the linkers due to the addition of uniquerestriction sites in the linker sequence to facilitate construction ofthe fusions.

In some embodiments the linkers comprise sequences selected from thegroup of formulas: (Gly₃Ser)_(n), (Gly₄Ser)_(n), (Gly₅Ser)_(n),(Gly_(n)Ser)_(n) or (AlaGlySer)_(n) where n is an integer. One exampleof a highly-flexible linker is the (GlySer)-rich spacer region presentwithin the pill protein of the filamentous bacteriophages, e.g.bacteriophages M13 or fd (Schaller, et al., 1975). This region providesa long, flexible spacer region between two domains of the pill surfaceprotein. Also included are linkers in which an endopeptidase recognitionsequence is included. Such a cleavage site may be valuable to separatethe individual components of the fusion to determine if they areproperly folded and active in vitro. Examples of various endopeptidasesinclude, but are not limited to, Plasmin, Enterokinase, Kallikerin,Urokinase, Tissue Plasminogen activator, clostripain, Chymosin,Collagenase, Russell's Viper Venom Protease, Postproline cleavageenzyme, V8 protease, Thrombin and factor Xa. In some embodiments thelinker comprises the amino acids EEKKN (SEQ ID NO: 215) from themulti-gene expression vehicle (MGEV), which is cleaved by vacuolarproteases as disclosed in US Patent Application Publication Number US2007/0277263. In other embodiments, peptide linker segments from thehinge region of heavy chain immunoglobulins IgG, IgA, IgM, IgD or IgEprovide an angular relationship between the attached polypeptides.Especially useful are those hinge regions where the cysteines arereplaced with serines. Linkers of the present disclosure includesequences derived from murine IgG gamma 2b hinge region in which thecysteines have been changed to serines. The fusion proteins are notlimited by the form, size or number of linker sequences employed and theonly requirement of the linker is that functionally it does notinterfere adversely with the folding and function of the individualmolecules of the fusion.

In another aspect chimeric insecticidal polypeptides are provided thatare created through joining two or more portions of insecticidalpolypeptides genes of disclosure, which originally encoded separateinsecticidal proteins to create a chimeric gene. The translation of thechimeric gene results in a single chimeric insecticidal polypeptide withregions, motifs or domains derived from each of the originalpolypeptides.

It is recognized that DNA sequences may be altered by various methods,and that these alterations may result in DNA sequences encoding proteinswith amino acid sequences different than that encoded by the wild-type(or native) pesticidal protein. In some embodiments an insecticidalpolypeptide of the disclosure may be altered in various ways includingamino acid substitutions, deletions, truncations and insertions of oneor more amino acids, including up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or more aminoacid substitutions, deletions and/or insertions or combinations thereofcompared to any one of SEQ ID NO: 1-SEQ ID NO: 107, and SEQ ID NO:232-SEQ ID NO: 245. In some embodiments an insecticidal polypeptide ofthe disclosure comprises the deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15 or more amino acids from the N-terminus and/orC-terminus of the insecticidal polypeptide of the disclosure.

Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of an insecticidal polypeptide ofthe disclosure can be prepared by mutations in the DNA. This may also beaccomplished by one of several forms of mutagenesis and/or in directedevolution. In some aspects, the changes encoded in the amino acidsequence will not substantially affect the function of the protein. Suchvariants will possess the desired pesticidal activity. However, it isunderstood that the ability of an insecticidal polypeptide of thedisclosure to confer pesticidal activity may be improved by the use ofsuch techniques upon the compositions of this disclosure.

For example, conservative amino acid substitutions may be made at one ormore, predicted, nonessential amino acid residues. A “nonessential”amino acid residue is a residue that can be altered from the wild-typesequence of an insecticidal polypeptide of the disclosure withoutaltering the biological activity. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include: amino acids with basic side chains (e.g., lysine,arginine, histidine); acidic side chains (e.g., aspartic acid, glutamicacid); polar, negatively charged residues and their amides (e.g.,aspartic acid, asparagine, glutamic acid, glutamine; uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine); small aliphatic, nonpolar or slightly polarresidues (e.g., Alanine, serine, threonine, proline, glycine); nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan); large aliphatic, nonpolarresidues (e.g., methionine, leucine, isoleucine, valine, cysteine);beta-branched side chains (e.g., threonine, valine, isoleucine);aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine); large aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan).

Amino acid substitutions may be made in nonconserved regions that retainfunction. In general, such substitutions would not be made for conservedamino acid residues or for amino acid residues residing within aconserved motif, where such residues are essential for protein activity.Examples of residues that are conserved and that may be essential forprotein activity include, for example, residues that are identicalbetween all proteins contained in an alignment of similar or relatedtoxins to the sequences of the embodiments (e.g., residues that areidentical in an alignment of homologs). Examples of residues that areconserved but that may allow conservative amino acid substitutions andstill retain activity include, for example, residues that have onlyconservative substitutions between all proteins contained in analignment of similar or related toxins to the sequences of theembodiments (e.g., residues that have only conservative substitutionsbetween all proteins contained in the alignment of the homologs).However, one of skill in the art would understand that functionalvariants may have minor conserved or nonconserved alterations in theconserved residues. Guidance as to appropriate amino acid substitutionsthat do not affect biological activity of the protein of interest may befound in the model of Dayhoff, et al., (1978) Atlas of Protein Sequenceand Structure (Natl. Biomed. Res. Found., Washington, D.C.), hereinincorporated by reference.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, (1982) J Mol Biol.157(1):105-32). It is accepted that the relative hydropathic characterof the amino acid contributes to the secondary structure of theresultant protein, which in turn defines the interaction of the proteinwith other molecules, for example, enzymes, substrates, receptors, DNA,antibodies, antigens, and the like.

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. Each amino acid has beenassigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte and Doolittle, ibid). These are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9) and arginine(−4.5). In making such changes, the substitution of amino acids whosehydropathic indices are within +2 is preferred, those which are within+1 are particularly preferred, and those within +0.5 are even moreparticularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, states that the greatest local average hydrophilicity ofa protein, as governed by the hydrophilicity of its adjacent aminoacids, correlates with a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0.+0.1); glutamate (+3.0.+0.1); serine(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine(−0.4); proline (−0.5.+0.1); alanine (−0.5); histidine (−0.5); cysteine(−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine(−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

Alternatively, alterations may be made to the protein sequence of manyproteins at the amino or carboxy terminus without substantiallyaffecting activity. This can include insertions, deletions oralterations introduced by modern molecular methods, such as PCR,including PCR amplifications that alter or extend the protein codingsequence by virtue of inclusion of amino acid encoding sequences in theoligonucleotides utilized in the PCR amplification. Alternatively, theprotein sequences added can include entire protein-coding sequences,such as those used commonly in the art to generate protein fusions. Suchfusion proteins are often used to (1) increase expression of a proteinof interest (2) introduce a binding domain, enzymatic activity orepitope to facilitate either protein purification, protein detection orother experimental uses known in the art (3) target secretion ortranslation of a protein to a subcellular organelle, such as theperiplasmic space of Gram-negative bacteria, mitochondria orchloroplasts of plants or the endoplasmic reticulum of eukaryotic cells,the latter of which often results in glycosylation of the protein.

Variant nucleotide and amino acid sequences of the disclosure alsoencompass sequences derived from mutagenic and recombinogenic proceduressuch as DNA shuffling. With such a procedure, one or more differentinsecticidal polypeptide of the disclosure coding regions can be used tocreate a new insecticidal polypeptide of the disclosure possessing thedesired properties. In this manner, libraries of recombinantpolynucleotides are generated from a population of related sequencepolynucleotides comprising sequence regions that have substantialsequence identity and can be homologously recombined in vitro or invivo. For example, using this approach, sequence motifs encoding adomain of interest may be shuffled between a pesticidal gene and otherknown pesticidal genes to obtain a new gene coding for a protein with animproved property of interest, such as an increased insecticidalactivity. Strategies for such DNA shuffling are known in the art. See,for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer, (1994) Nature 370:389-391; Crameri, et al., (1997) NatureBiotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol. 272:336-347;Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri,et al., (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and5,837,458.

Domain swapping or shuffling is another mechanism for generating alteredinsecticidal polypeptides of the disclosure. Domains may be swappedbetween insecticidal polypeptides of the disclosure, resulting in hybridor chimeric toxins with improved insecticidal activity or targetspectrum. Methods for generating recombinant proteins and testing themfor pesticidal activity are well known in the art (see, for example,Naimov, et al., (2001) Appl. Environ. Microbiol. 67:5328-5330; de Maagd,et al., (1996) Appl. Environ. Microbiol. 62:1537-1543; Ge, et al.,(1991) J. Biol. Chem. 266:17954-17958; Schnepf, et al., (1990) J. Biol.Chem. 265:20923-20930; Rang, et al., 91999) Appl. Environ. Microbiol.65:2918-2925).

Alignment of homologs of the insectidal polypeptide (FIGS. 1, 2, 3, 4,5, 6, 7 & 8) allows for identification of residues that are highlyconserved among homologs in these families.

Compositions

Compositions comprising the insecticidal polypeptides of the presentdisclosure are also envisioned. Compositions comprising a PIP-45-1polypeptide of the disclosure and a PIP-45-2 polypeptide of thedisclosure are contemplated. In some embodiments the compositionscomprise an insecticidally effective concentration of a PIP-45-1polypeptide of the disclosure and a PIP-45-2 polypeptide of thedisclosure. Compositions comprising a PIP-64-1 polypeptide of thedisclosure and a PIP-64-2 polypeptide of the disclosure arecontemplated. In some embodiments the compositions comprise aninsecticidally effective concentration of a PIP-64-1 polypeptide of thedisclosure and a PIP-64-2 polypeptide of the disclosure. Compositionscomprising a PIP-74-1 polypeptide of the disclosure and a PIP-74-2polypeptide of the disclosure are contemplated. In some embodiments thecompositions comprise an insecticidally effective concentration of aPIP-74-1 polypeptide of the disclosure and a PIP-74-2 polypeptide of thedisclosure. Compositions comprising a PIP-75 polypeptide of thedisclosure are contemplated. In some embodiments the compositionscomprise an insecticidally effective concentration of a PIP-75polypeptide of the disclosure. Compositions comprising a PIP-77polypeptide of the disclosure are contemplated. In some embodiments thecompositions comprise an insecticidally effective concentration of aPIP-77 polypeptide of the disclosure. In some embodiments thecomposition further comprises an agriculturally acceptable carrier.

Antibodies

Antibodies to an insecticidal polypeptide of the disclosure of theembodiments or to variants or fragments thereof are also encompassed.The antibodies of the disclosure include polyclonal and monoclonalantibodies as well as fragments thereof which retain their ability tobind to insecticidal proteins found in the insect gut. An antibody,monoclonal antibody or fragment thereof is said to be capable of bindinga molecule if it is capable of specifically reacting with the moleculeto thereby bind the molecule to the antibody, monoclonal antibody orfragment thereof. The term “antibody” (Ab) or “monoclonal antibody”(Mab) is meant to include intact molecules as well as fragments orbinding regions or domains thereof (such as, for example, Fab andF(ab).sub.2 fragments) which are capable of binding hapten. Suchfragments are typically produced by proteolytic cleavage, such as papainor pepsin. Alternatively, hapten-binding fragments can be producedthrough the application of recombinant DNA technology or throughsynthetic chemistry. Methods for the preparation of the antibodies ofthe present disclosure are generally known in the art. For example, see,Antibodies, A Laboratory Manual, Ed Harlow and David Lane (eds.) ColdSpring Harbor Laboratory, N.Y. (1988), as well as the references citedtherein. Standard reference works setting forth the general principlesof immunology include: Klein, J. Immunology: The Science of Cell-NoncellDiscrimination, John Wiley & Sons, N.Y. (1982); Dennett, et al.,Monoclonal Antibodies, Hybridoma: A New Dimension in BiologicalAnalyses, Plenum Press, N.Y. (1980) and Campbell, “Monoclonal AntibodyTechnology,” In Laboratory Techniques in Biochemistry and MolecularBiology, Vol. 13, Burdon, et al., (eds.), Elsevier, Amsterdam (1984).See also, U.S. Pat. Nos. 4,196,265; 4,609,893; 4,713,325; 4,714,681;4,716,111; 4,716,117 and 4,720,459. Antibodies to the insecticidalpolypeptides of the disclosure or antigen-binding portions thereof canbe produced by a variety of techniques, including conventionalmonoclonal antibody methodology, for example the standard somatic cellhybridization technique of Kohler and Milstein, (1975) Nature 256:495.Other techniques for producing monoclonal antibody can also be employedsuch as viral or oncogenic transformation of B lymphocytes. An animalsystem for preparing hybridomas is a murine system. Immunizationprotocols and techniques for isolation of immunized splenocytes forfusion are known in the art. Fusion partners (e.g., murine myelomacells) and fusion procedures are also known. The antibody and monoclonalantibodies of the disclosure can be prepared by utilizing aninsecticidal polypeptide of the disclosure as antigens.

A kit for detecting the presence of an insecticidal polypeptide of thedisclosure or detecting the presence of a nucleotide sequence encodingan insecticidal polypeptide of the disclosure, in a sample is provided.In one embodiment, the kit provides antibody-based reagents fordetecting the presence of an insecticidal polypeptide of the disclosurein a tissue sample. In another embodiment, the kit provides labelednucleic acid probes useful for detecting the presence of one or morepolynucleotides encoding an insecticidal polypeptide(s) of thedisclosure. The kit is provided along with appropriate reagents andcontrols for carrying out a detection method, as well as instructionsfor use of the kit.

Receptor Identification and Isolation

Receptors to the insecticidal polypeptide of the embodiments or tovariants or fragments thereof, are also encompassed. Methods foridentifying receptors are well known in the art (see, Hofmann, et. al.,(1988) Eur. J. Biochem. 173:85-91; Gill, et al., (1995) J. Biol. Chem.27277-27282) can be employed to identify and isolate the receptor thatrecognizes the insecticidal polypeptides of the disclosure using thebrush-border membrane vesicles from susceptible insects. In addition tothe radioactive labeling method listed in the cited literature,insecticidal polypeptide can be labeled with fluorescent dye and othercommon labels such as streptavidin. Brush-border membrane vesicles(BBMV) of susceptible insects such as soybean looper and stink bugs canbe prepared according to the protocols listed in the references andseparated on SDS-PAGE gel and blotted on suitable membrane. Labeledinsecticidal polypeptides of the disclosure can be incubated withblotted membrane of BBMV and labeled the insecticidal polypeptides ofthe disclosure can be identified with the labeled reporters.Identification of protein band(s) that interact with the insecticidalpolypeptides of the disclosure can be detected by N-terminal amino acidgas phase sequencing or mass spectrometry based protein identificationmethod (Patterson, (1998) 10.22, 1-24, Current Protocol in MolecularBiology published by John Wiley & Son Inc). Once the protein isidentified, the corresponding gene can be cloned from genomic DNA orcDNA library of the susceptible insects and binding affinity can bemeasured directly with the insecticidal polypeptides of the disclosure.Receptor function for insecticidal activity by the insecticidalpolypeptides of he disclosure can be verified by accomplished by RNAitype of gene knock out method (Rajagopal, et al., (2002) J. Biol. Chem.277:46849-46851).

Nucleotide Constructs, Expression Cassettes and Vectors

The use of the term “nucleotide constructs” herein is not intended tolimit the embodiments to nucleotide constructs comprising DNA. Those ofordinary skill in the art will recognize that nucleotide constructsparticularly polynucleotides and oligonucleotides composed ofribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides may also be employed in the methods disclosedherein. The nucleotide constructs, nucleic acids, and nucleotidesequences of the embodiments additionally encompass all complementaryforms of such constructs, molecules, and sequences. Further, thenucleotide constructs, nucleotide molecules, and nucleotide sequences ofthe embodiments encompass all nucleotide constructs, molecules, andsequences which can be employed in the methods of the embodiments fortransforming plants including, but not limited to, those comprised ofdeoxyribonucleotides, ribonucleotides, and combinations thereof. Suchdeoxyribonucleotides and ribonucleotides include both naturallyoccurring molecules and synthetic analogues. The nucleotide constructs,nucleic acids, and nucleotide sequences of the embodiments alsoencompass all forms of nucleotide constructs including, but not limitedto, single-stranded forms, double-stranded forms, hairpins,stem-and-loop structures and the like.

A further embodiment relates to a transformed organism such as anorganism selected from plant and insect cells, bacteria, yeast,baculovirus, protozoa, nematodes and algae. The transformed organismcomprises a DNA molecule of the embodiments, an expression cassettecomprising the DNA molecule or a vector comprising the expressioncassette, which may be stably incorporated into the genome of thetransformed organism.

The sequences of the embodiments are provided in DNA constructs forexpression in the organism of interest. The construct will include 5′and 3′ regulatory sequences operably linked to a sequence of theembodiments. The term “operably linked” as used herein refers to afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand where necessary to join two protein coding regions in the samereading frame. The construct may additionally contain at least oneadditional gene to be cotransformed into the organism. Alternatively,the additional gene(s) can be provided on multiple DNA constructs.

Such a DNA construct is provided with a plurality of restriction sitesfor insertion of the insecticidal polypeptide gene sequence to be underthe transcriptional regulation of the regulatory regions. The DNAconstruct may additionally contain selectable marker genes.

The DNA construct will generally include in the 5′ to 3′ direction oftranscription: a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the embodiments, and atranscriptional and translational termination region (i.e., terminationregion) functional in the organism serving as a host. Thetranscriptional initiation region (i.e., the promoter) may be native,analogous, foreign or heterologous to the host organism and/or to thesequence of the embodiments. Additionally, the promoter may be thenatural sequence or alternatively a synthetic sequence. The term“foreign” as used herein indicates that the promoter is not found in thenative organism into which the promoter is introduced. Where thepromoter is “foreign” or “heterologous” to the sequence of theembodiments, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked sequence of theembodiments. As used herein, a chimeric gene comprises a coding sequenceoperably linked to a transcription initiation region that isheterologous to the coding sequence. Where the promoter is a native ornatural sequence, the expression of the operably linked sequence isaltered from the wild-type expression, which results in an alteration inphenotype.

In some embodiments the DNA construct may also include a transcriptionalenhancer sequence. As used herein, the term an “enhancer” refers to aDNA sequence which can stimulate promoter activity, and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Various enhancers areknown in the art including for example, introns with gene expressionenhancing properties in plants (US Patent Application Publication Number2009/0144863, the ubiquitin intron (i.e., the maize ubiquitin intron 1(see, for example, NCBI sequence S94464; Christensen and Quail (1996)Transgenic Res. 5:213-218; Christensen et al. (1992) Plant MolecularBiology 18:675-689)), the omega enhancer or the omega prime enhancer(Gallie, et al., (1989) Molecular Biology of RNA ed. Cech (Liss, N.Y.)237-256 and Gallie, et al., (1987) Gene 60:217-25), the CaMV 35Senhancer (see, e.g., Benfey, et al., (1990) EMBO J. 9:1685-96), themaize Adhl intron (Kyozuka et al. (1991) Mol. Gen. Genet. 228:40-48;Kyozuka et al. (1990) Maydica 35:353-357) and the enhancers of U.S. Pat.No. 7,803,992 may also be used, each of which is incorporated byreference. The above list of transcriptional enhancers is not meant tobe limiting. Any appropriate transcriptional enhancer can be used in theembodiments.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host or may be derived from another source(i.e., foreign or heterologous to the promoter, the sequence ofinterest, the plant host or any combination thereof).

Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau, et al, (1991) Mol. Gen. Genet.262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al., (1991)Genes Dev. 5:141-149; Mogen, et al, (1990) Plant Cell 2:1261-1272;Munroe, et al., (1990) Gene 91:151-158; Ballas, et al., (1989) NucleicAcids Res. 17:7891-7903 and Joshi, et al., (1987) Nucleic Acid Res.15:9627-9639.

Where appropriate, a nucleic acid may be optimized for increasedexpression in the host organism. Thus, where the host organism is aplant, the synthetic nucleic acids can be synthesized usingplant-preferred codons for improved expression. See, for example,Campbell and Gowri, (1990) Plant Physiol. 92:1-11 for a discussion ofhost-preferred codon usage. For example, although nucleic acid sequencesof the embodiments may be expressed in both monocotyledonous anddicotyledonous plant species, sequences can be modified to account forthe specific codon preferences and GC content preferences ofmonocotyledons or dicotyledons as these preferences have been shown todiffer (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, themaize-preferred codon for a particular amino acid may be derived fromknown gene sequences from maize. Maize codon usage for 28 genes frommaize plants is listed in Table 4 of Murray, et al, supra. Methods areavailable in the art for synthesizing plant-preferred genes. See, forexample, U.S. Pat. Nos. 5,380,831, and 5,436,391 and Murray, et al.,(1989) Nucleic Acids Res. 17:477-498, and Liu H et al. Mol Bio Rep37:677-684, 2010, herein incorporated by reference. A Zea maize codonusage table can be also found atkazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=4577, which can beaccessed using the www prefix. Table 2 shows a maize optimal codonanalysis (adapted from Liu H et al. Mol Bio Rep 37:677-684, 2010).

TABLE 2 Amino High Low Amino High Low Acid Codon Count RSCU Count RSCUAcid Codon Count RSCU Count RSCU Phe UUU 115 0.04 2,301 1.22 Ala GCU 6290.17 3,063 1.59 UUC* 5,269 1.96 1,485 0.78 GCC* 8,057 2.16 1,136 0.59Ser UCU 176 0.13 2,498 1.48 GCA 369 0.1 2,872 1.49 UCC* 3,489 2.48 1,0740.63 GCG* 5,835 1.57 630 0.33 UCA 104 0.07 2,610 1.54 Tyr UAU 71 0.041,632 1.22 UCG* 1,975 1.4 670 0.4 UAC* 3,841 1.96 1,041 0.78 AGU 77 0.051,788 1.06 His CAU 131 0.09 1,902 1.36 AGC* 2,617 1.86 1,514 0.89 CAC*2,800 1.91 897 0.64 Leu UUA 10 0.01 1,326 0.79 Cys UGU 52 0.04 1,2331.12 UUG 174 0.09 2,306 1.37 UGC* 2,291 1.96 963 0.88 CUU 223 0.11 2,3961.43 Gln CAA 99 0.05 2,312 1.04 CUC* 5,979 3.08 1,109 0.66 CAG* 3,5571.95 2,130 0.96 CUA 106 0.05 1,280 0.76 Arg CGU 153 0.12 751 0.74 CUG*5,161 2.66 1,646 0.98 CGC* 4,278 3.25 466 0.46 Pro CCU 427 0.22 1,9001.47 CGA 92 0.07 659 0.65 CCC* 3,035 1.59 601 0.47 CGG* 1,793 1.36 6310.62 CCA 311 0.16 2,140 1.66 AGA 83 0.06 1,948 1.91 CCG* 3,846 2.02 5130.4 AGG* 1,493 1.14 1,652 1.62 Ile AUU 138 0.09 2,388 1.3 Asn AAU 1310.07 3,074 1.26 AUC* 4,380 2.85 1,353 0.74 AAC* 3,814 1.93 1,807 0.74AUA 88 0.06 1,756 0.96 Lys AAA 130 0.05 3,215 0.98 Thr ACU 136 0.091,990 1.43 AAG* 5,047 1.95 3,340 1.02 ACC* 3,398 2.25 991 0.71 Asp GAU312 0.09 4,217 1.38 ACA 133 0.09 2,075 1.5 GAC* 6,729 1.91 1,891 0.62ACG* 2,378 1.57 495 0.36 Gly GGU 363 0.13 2,301 1.35 Val GUU 182 0.072,595 1.51 GGC* 7,842 2.91 1,282 0.75 GUC* 4,584 1.82 1,096 0.64 GGA 3970.15 2,044 1.19 GUA 74 0.03 1,325 0.77 GGG* 2,186 0.81 1,215 0.71 GUG*5,257 2.08 1,842 1.07 Glu GAA 193 0.06 4,080 1.1 GAG* 6,010 1.94 3,3070.9 Codon usage was compared using Chi squared contingency test toidentify optimal codons. Codons that occur significantly more often(P\0.01) are indicated with an asterisk.

A Glycine max codon usage table is shown in Table 3 and can also befound atkazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=3847&aa=1&style=N,which can be accessed using the www prefix.

TABLE 3 TTT F 21.2 (10493) TCT S 18.4  (9107) TTC F 21.2 (10487) TCC S12.9  (6409) TTA L 9.2  (4545) TCA S 15.6  (7712) TTG L 22.9 (11340) TCGS 4.8  (2397) CTT L 23.9 (11829) CCT P 18.9  (9358) CTC L 17.1  (8479)CCC P 10.1  (5010) CTA L 8.5  (4216) CCA P 19.1  (9461) CTG L 12.7 (6304) CCG P 4.7  (2312) ATT I 25.1 (12411) ACT T 17.1  (8490) ATC I16.3  (8071) ACC T 14.3  (7100) ATA I 12.9  (6386) ACA T 14.9  (7391)ATG M 22.7 (11218) ACG T 4.3  (2147) GTT V 26.1 (12911) GCT A 26.7(13201) GTC V 11.9  (5894) GCC A 16.2  (8026) GTA V 7.7  (3803) GCA A21.4 (10577) GTG V 21.4 (10610) GCG A 6.3  (3123) TAT Y 15.7  (7779) TGTC 8.1  (3995) TAC Y 14.9  (7367) TGC C 8.0  (3980) TAA * 0.9   (463)TGA * 1.0   (480) TAG * 0.5   (263) TGG W 13.0  (6412) CAT H 14.0 (6930) CGT R 6.6  (3291) CAC H 11.6  (5759) CGC R 6.2  (3093) CAA Q20.5 (10162) CGA R 4.1  (2018) CAG Q 16.2  (8038) CGG R 3.1  (1510) AATN 22.4 (11088) AGT S 12.6  (6237) AAC N 22.8 (11284) AGC S 11.3  (5594)AAA K 26.9 (13334) AGA R 14.8  (7337) AAG K 35.9 (17797) AGG R 13.3 (6574) GAT D 32.4 (16040) GGT G 20.9 (10353) GAC D 20.4 (10097) GGC G13.4  (6650) GAA E 33.2 (16438) GGA G 22.3 (11022) GAG E 33.2 (16426)GGG G 13.0  (6431)

In some embodiments the recombinant nucleic acid molecule encoding aninsecticidal polypeptide of the disclosure has maize optimized codons.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other well-characterized sequences that maybe deleterious to gene expression. The GC content of the sequence may beadjusted to levels average for a given cellular host, as calculated byreference to known genes expressed in the host cell. The term “hostcell” as used herein refers to a cell which contains a vector andsupports the replication and/or expression of the expression vector isintended. Host cells may be prokaryotic cells such as E. coli oreukaryotic cells such as yeast, insect, amphibian or mammalian cells ormonocotyledonous or dicotyledonous plant cells. An example of amonocotyledonous host cell is a maize host cell. When possible, thesequence is modified to avoid predicted hairpin secondary mRNAstructures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie,et al., (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus), human immunoglobulin heavy-chain binding protein (BiP) (Macejak,et al., (1991) Nature 353:90-94); untranslated leader from the coatprotein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al.,(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie,et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, N.Y.), pp.237-256) and maize chlorotic mottle virus leader (MCMV) (Lommel, et al.,(1991) Virology 81:382-385). See also, Della-Cioppa, et al., (1987)Plant Physiol. 84:965-968. Such constructs may also contain a “signalsequence” or “leader sequence” to facilitate co-translational orpost-translational transport of the peptide to certain intracellularstructures such as the chloroplast (or other plastid), endoplasmicreticulum or Golgi apparatus.

“Signal sequence” as used herein refers to a sequence that is known orsuspected to result in cotranslational or post-translational peptidetransport across the cell membrane. In eukaryotes, this typicallyinvolves secretion into the Golgi apparatus, with some resultingglycosylation. Insecticidal toxins of bacteria are often synthesized asprotoxins, which are protolytically activated in the gut of the targetpest (Chang, (1987) Methods Enzymol. 153:507-516). In some embodiments,the signal sequence is located in the native sequence or may be derivedfrom a sequence of the embodiments. “Leader sequence” as used hereinrefers to any sequence that when translated, results in an amino acidsequence sufficient to trigger co-translational transport of the peptidechain to a subcellular organelle. Thus, this includes leader sequencestargeting transport and/or glycosylation by passage into the endoplasmicreticulum, passage to vacuoles, plastids including chloroplasts,mitochondria, and the like. Nuclear-encoded proteins targeted to thechloroplast thylakoid lumen compartment have a characteristic bipartitetransit peptide, composed of a stromal targeting signal peptide and alumen targeting signal peptide. The stromal targeting information is inthe amino-proximal portion of the transit peptide. The lumen targetingsignal peptide is in the carboxyl-proximal portion of the transitpeptide, and contains all the information for targeting to the lumen.Recent research in proteomics of the higher plant chloroplast hasachieved in the identification of numerous nuclear-encoded lumenproteins (Kieselbach et al. FEBS LETT 480:271-276, 2000; Peltier et al.Plant Cell 12:319-341, 2000; Bricker et al. Biochim. Biophys Acta1503:350-356, 2001), the lumen targeting signal peptide of which canpotentially be used in accordance with the present disclosure. About 80proteins from Arabidopsis, as well as homologous proteins from spinachand garden pea, are reported by Kieselbach et al., PhotosynthesisResearch, 78:249-264, 2003. In particular, Table 2 of this publication,which is incorporated into the description herewith by reference,discloses 85 proteins from the chloroplast lumen, identified by theiraccession number (see also US Patent Application Publication2009/09044298). In addition, the recently published draft version of therice genome (Goff et al, Science 296:92-100, 2002) is a suitable sourcefor lumen targeting signal peptide which may be used in accordance withthe present disclosure.

Suitable chloroplast transit peptides (CTP) are well known to oneskilled in the art also include chimeric CTPs comprising but not limitedto, an N-terminal domain, a central domain or a C-terminal domain from aCTP from Oryza sativa 1-deoxy-D xyulose-5-Phosphate Synthase Oryzasativa-Superoxide dismutase Oryza sativa-soluble starch synthase Oryzasativa-NADP-dependent Malic acid enzyme Oryzasativa-Phospho-2-dehydro-3-deoxyheptonate Aldolase 2 Oryzasativa-L-Ascorbate peroxidase 5 Oryza sativa-Phosphoglucan waterdikinase, Zea mays ssRUBISCO, Zea mays-beta-glucosidase, Zea mays-Malatedehydrogenase, Zea mays Thioredoxin M-type (US Patent ApplicationPublication 2012/0304336). Chloroplast transit peptides of US PatentPublications US20130205440A1, US20130205441A1 and U520130210114A1.

The insecticidal polypeptide gene to be targeted to the chloroplast maybe optimized for expression in the chloroplast to account fordifferences in codon usage between the plant nucleus and this organelle.In this manner, the nucleic acids of interest may be synthesized usingchloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831,herein incorporated by reference.

In preparing the expression cassette, the various DNA fragments may bemanipulated so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used in the practice of the embodiments.The promoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-preferred, inducible orother promoters for expression in the host organism. Suitableconstitutive promoters for use in a plant host cell include, forexample, the core promoter of the Rsyn7 promoter and other constitutivepromoters disclosed in WO 1999/43838 and U.S. Pat. No. 6,072,050; thecore CaMV 35S promoter (Odell, et al., (1985) Nature 313:810-812); riceactin (McElroy, et al., (1990) Plant Cell 2:163-171); ubiquitin(Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 andChristensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, etal., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten, et al., (1984)EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026) and thelike. Other constitutive promoters include, for example, those discussedin U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142 and 6,177,611. Suitable constitutivepromoters also include promoters that have strong expression in nearlyall tissues but have low expression in pollen, including but not limitedto: Banana Streak Virus (Acuminata Yunnan) promoters (BSV(AY)) disclosedin U.S. Pat. No. 8,338,662; Banana Streak Virus (Acuminata Vietnam)promoters (BSV(AV)) disclosed in U.S. Pat. No. 8,350,121; and BananaStreak Virus (Mysore) promoters (BSV(MYS)) disclosed in U.S. Pat. No.8,395,022.

Depending on the desired outcome, it may be beneficial to express thegene from an inducible promoter. Of particular interest for regulatingthe expression of the nucleotide sequences of the embodiments in plantsare wound-inducible promoters. Such wound-inducible promoters, mayrespond to damage caused by insect feeding, and include potatoproteinase inhibitor (pin II) gene (Ryan, (1990) Ann. Rev. Phytopath.28:425-449; Duan, et al., (1996) Nature Biotechnology 14:494-498); wun1and wun2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford, et al.,(1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl, et al., (1992)Science 225:1570-1573); WIP1 (Rohmeier, et al, (1993) Plant Mol. Biol.22:783-792; Eckelkamp, et al., (1993) FEBS Letters 323:73-76); MPI gene(Corderok, et al., (1994) Plant J. 6(2):141-150) and the like, hereinincorporated by reference.

Additionally, pathogen-inducible promoters may be employed in themethods and nucleotide constructs of the embodiments. Suchpathogen-inducible promoters include those from pathogenesis-relatedproteins (PR proteins), which are induced following infection by apathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase,chitinase, etc. See, for example, Redolfi, et al., (1983) Neth. J. PlantPathol. 89:245-254; Uknes, et al., (1992) Plant Cell 4: 645-656 and VanLoon, (1985) Plant Mol. Virol. 4:111-116. See also, WO 1999/43819,herein incorporated by reference.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau, et al., (1987) PlantMol. Biol. 9:335-342; Matton, et al., (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch, et al., (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch, et al., (1988) Mol. Gen. Genet. 2:93-98 andYang, (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen,et al., (1996) Plant J. 10:955-966; Zhang, et al., (1994) Proc. Natl.Acad. Sci. USA 91:2507-2511; Warner, et al., (1993) Plant J. 3:191-201;Siebertz, et al., (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible) and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero, et al., (1992) Physiol. Mol. Plant Path. 41:189-200).

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression or a chemical-repressible promoter, where application ofthe chemical represses gene expression. Chemical-inducible promoters areknown in the art and include, but are not limited to, the maize In2-2promoter, which is activated by benzenesulfonamide herbicide safeners,the maize GST promoter, which is activated by hydrophobic electrophiliccompounds that are used as pre-emergent herbicides, and the tobaccoPR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena, et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis, et al., (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237 and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhancedinsecticidal polypeptide expression within a particular plant tissue.Tissue-preferred promoters include those discussed in Yamamoto, et al.,(1997) Plant J. 12(2)255-265; Kawamata, et al., (1997) Plant CellPhysiol. 38(7):792-803; Hansen, et al., (1997) Mol. Gen Genet.254(3):337-343; Russell, et al., (1997) Transgenic Res. 6(2):157-168;Rinehart, et al., (1996) Plant Physiol. 112(3):1331-1341; Van Camp, etal., (1996) Plant Physiol. 112(2):525-535; Canevascini, et al., (1996)Plant Physiol. 112(2):513-524; Yamamoto, et al., (1994) Plant CellPhysiol. 35(5):773-778; Lam, (1994) Results Probl. Cell Differ.20:181-196; Orozco, et al., (1993) Plant Mol Biol. 23(6):1129-1138;Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 andGuevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Such promoters canbe modified, if necessary, for weak expression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kwon, et al., (1994)Plant Physiol. 105:357-67; Yamamoto, et al., (1994) Plant Cell Physiol.35(5):773-778; Gotor, et al., (1993) Plant J. 3:509-18; Orozco, et al.,(1993) Plant Mol. Biol. 23(6):1129-1138 and Matsuoka, et al., (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred or root-specific promoters are known and can be selectedfrom the many available from the literature or isolated de novo fromvarious compatible species. See, for example, Hire, et al., (1992) PlantMol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetasegene); Keller and Baumgartner, (1991) Plant Cell 3(10):1051-1061(root-specific control element in the GRP 1.8 gene of French bean);Sanger, et al., (1990) Plant Mol. Biol. 14(3):433-443 (root-specificpromoter of the mannopine synthase (MAS) gene of Agrobacteriumtumefaciens) and Miao, et al., (1991) Plant Cell 3(1):11-22 (full-lengthcDNA clone encoding cytosolic glutamine synthetase (GS), which isexpressed in roots and root nodules of soybean). See also, Bogusz, etal., (1990) Plant Cell 2(7):633-641, where two root-specific promotersisolated from hemoglobin genes from the nitrogen-fixing nonlegumeParasponia andersonii and the related non-nitrogen-fixing nonlegumeTrema tomentosa are described. The promoters of these genes were linkedto a 3-glucuronidase reporter gene and introduced into both thenonlegume Nicotiana tabacum and the legume Lotus corniculatus, and inboth instances root-specific promoter activity was preserved. Leach andAoyagi, (1991) describe their analysis of the promoters of the highlyexpressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes(see, Plant Science (Limerick) 79(1):69-76). They concluded thatenhancer and tissue-preferred DNA determinants are dissociated in thosepromoters. Teeri, et al., (1989) used gene fusion to lacZ to show thatthe Agrobacterium T-DNA gene encoding octopine synthase is especiallyactive in the epidermis of the root tip and that the TR2′ gene is rootspecific in the intact plant and stimulated by wounding in leaf tissue,an especially desirable combination of characteristics for use with aninsecticidal or larvicidal gene (see, EMBO J. 8(2):343-350). The TR1′gene fused to nptII (neomycin phosphotransferase II) showed similarcharacteristics. Additional root-preferred promoters include theVfENOD-GRP3 gene promoter (Kuster, et al., (1995) Plant Mol. Biol.29(4):759-772) and rolB promoter (Capana, et al., (1994) Plant Mol.Biol. 25(4):681-691. See also, U.S. Pat. Nos. 5,837,876; 5,750,386;5,633,363; 5,459,252; 5,401,836; 5,110,732 and 5,023,179. Arabidopsisthaliana root-preferred regulatory sequences are disclosed in US PatentApplication US20130117883. Root-preferred sorghum (Sorghum bicolor) RCc3promoters are disclosed in US Patent Application US20120210463.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See, Thompson, et al., (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); and milps(myo-inositol-1-phosphate synthase) (see, U.S. Pat. No. 6,225,529,herein incorporated by reference). Gamma-zein and Glb-1 areendosperm-specific promoters. For dicots, seed-specific promotersinclude, but are not limited to, Kunitz trypsin inhibitor 3 (KTi3)(Jofuku and Goldberg, (1989) Plant Cell 1:1079-1093), bean β-phaseolin,napin, β-conglycinin, glycinin 1, soybean lectin, cruciferin, and thelike. For monocots, seed-specific promoters include, but are not limitedto, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken1, shrunken 2, globulin 1, etc. See also, WO 2000/12733, whereseed-preferred promoters from end1 and end2 genes are disclosed; hereinincorporated by reference. In dicots, seed specific promoters includebut are not limited to seed coat promoter from Arabidopsis, pBAN; andthe early seed promoters from Arabidopsis, p26, p63, and p63tr (U.S.Pat. Nos. 7,294,760 and 7,847,153). A promoter that has “preferred”expression in a particular tissue is expressed in that tissue to agreater degree than in at least one other plant tissue. Sometissue-preferred promoters show expression almost exclusively in theparticular tissue.

Where low level expression is desired, weak promoters will be used.Generally, the term “weak promoter” as used herein refers to a promoterthat drives expression of a coding sequence at a low level. By low levelexpression at levels of about 1/1000 transcripts to about 1/100,000transcripts to about 1/500,000 transcripts is intended. Alternatively,it is recognized that the term “weak promoters” also encompassespromoters that drive expression in only a few cells and not in others togive a total low level of expression. Where a promoter drives expressionat unacceptably high levels, portions of the promoter sequence can bedeleted or modified to decrease expression levels.

Such weak constitutive promoters include, for example the core promoterof the Rsyn7 promoter (WO 1999/43838 and U.S. Pat. No. 6,072,050), thecore 35S CaMV promoter, and the like. Other constitutive promotersinclude, for example, those disclosed in U.S. Pat. Nos. 5,608,149;5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463;5,608,142 and 6,177,611, herein incorporated by reference.

The above list of promoters is not meant to be limiting. Any appropriatepromoter can be used in the embodiments.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glufosinate ammonium, bromoxynil, imidazolinones and2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitableselectable marker genes include, but are not limited to, genes encodingresistance to chloramphenicol (Herrera Estrella, et al., (1983) EMBO J.2:987-992); methotrexate (Herrera Estrella, et al., (1983) Nature303:209-213 and Meijer, et al., (1991) Plant Mol. Biol. 16:807-820);streptomycin (Jones, et al., (1987) Mol. Gen. Genet. 210:86-91);spectinomycin (Bretagne-Sagnard, et al., (1996) Transgenic Res.5:131-137); bleomycin (Hille, et al., (1990) Plant Mol. Biol.7:171-176); sulfonamide (Guerineau, et al., (1990) Plant Mol. Biol.15:127-136); bromoxynil (Stalker, et al., (1988) Science 242:419-423);glyphosate (Shaw, et al., (1986) Science 233:478-481 and U.S. patentapplication Ser. Nos. 10/004,357 and 10/427,692); phosphinothricin(DeBlock, et al., (1987) EMBO J. 6:2513-2518). See generally, Yarranton,(1992) Curr. Opin. Biotech. 3:506-511; Christopherson, et al., (1992)Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao, et al., (1992) Cell71:63-72; Reznikoff, (1992) Mol. Microbiol. 6:2419-2422; Barkley, etal., (1980) in The Operon, pp. 177-220; Hu, et al., (1987) Cell48:555-566; Brown, et al., (1987) Cell 49:603-612; Figge, et al., (1988)Cell 52:713-722; Deuschle, et al., (1989) Proc. Natl. Acad. Sci. USA86:5400-5404; Fuerst, et al., (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle, et al., (1990) Science 248:480-483; Gossen,(1993) Ph.D. Thesis, University of Heidelberg; Reines, et al., (1993)Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow, et al., (1990) Mol.Cell. Biol. 10:3343-3356; Zambretti, et al., (1992) Proc. Natl. Acad.Sci. USA 89:3952-3956; Baim, et al., (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman, (1989) Topics Mol. Struc. Biol. 10:143-162;Degenkolb, et al., (1991) Antimicrob. Agents Chemother. 35:1591-1595;Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; Bonin, (1993)Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc.Natl. Acad. Sci. USA 89:5547-5551; Oliva, et al., (1992) Antimicrob.Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook ofExperimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin) and Gill,et al., (1988) Nature 334:721-724. Such disclosures are hereinincorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the embodiments.

DNA Constructs

DNA constructs comprising a polynucleotide encoding an insecticidalpolypeptide of the disclosure are encompassed. In some embodiments theDNA construct comprises a polynucleotide encoding a PIP-45-1 polypeptideoperably linked to a heterologous regulatory element. In someembodiments the DNA construct comprises the polynucleotide of SEQ ID NO:108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO:126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 218, SEQ ID NO:220 or SEQ ID NO: 222 that encodes the PIP-45-1 polypeptide of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29,SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ IDNO: 49, SEQ ID NO: 51, SEQ ID NO: 232, SEQ ID NO: 234 and SEQ ID NO:236, respectively. In some embodiments the DNA construct comprises thepolynucleotide of SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 126, SEQ IDNO: 128, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138,SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ IDNO: 152, SEQ ID NO: 220 or SEQ ID NO: 222, that encodes the PIP-45-1polypeptide of SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ IDNO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45, SEQID NO: 234 and SEQ ID NO: 236, respectively. In some embodiments the DNAconstruct comprises a non-genomic nucleic acid molecule encoding thePIP-45-1 polypeptide. In some embodiments the DNA construct comprises apolynucleotide encoding a PIP-45-1 polypeptide sufficiently homologousto the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ IDNO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 232,SEQ ID NO: 234 or SEQ ID NO: 236 and which has insecticidal activity. Insome embodiments the DNA construct comprises a polynucleotide encoding aPIP-45-1 polypeptide sufficiently homologous to the amino acid sequenceof SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO:23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ IDNO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 234 orSEQ ID NO: 236 and which has insecticidal activity. “Sufficientlyhomologous” is used herein to refer to an amino acid sequence that hasat least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homologycompared to a reference sequence using one of the alignment programsdescribed herein using standard parameters. One of skill in the art willrecognize that these values can be appropriately adjusted to determinecorresponding homology of proteins taking into account amino acidsimilarity and the like. In some embodiments the sequence homology isagainst the full length sequence of the PIP-45-1 polypeptide.

In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-45-1 polypeptide having at least about 50%, 55%, 60%,65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or greater sequence identity compared to SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21,SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ IDNO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQID NO: 51, SEQ ID NO: 232, SEQ ID NO: 234 or SEQ ID NO: 236 and whichhas insecticidal activity. In some embodiments the DNA constructcomprises a polynucleotide encoding a PIP-45-1 polypeptide having atleast about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identitycompared to SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21,SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45, SEQ IDNO: 234 or SEQ ID NO: 236 and which has insecticidal activity.

In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-45-1 polypeptide having at least 99.1% or greatersequence identity compared to SEQ ID NO: 1. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-45-1 polypeptidehaving at least 99.4% or greater sequence identity compared to SEQ IDNO: 17. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-45-1 polypeptide having at least 99.6% or greatersequence identity compared to SEQ ID NO: 19. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-45-1 polypeptidehaving at least 87% or greater sequence identity compared to SEQ ID NO:21. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-45-1 polypeptide having at least 88% or greater sequenceidentity compared to SEQ ID NO: 23. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-45-1 polypeptidehaving at least 99.1% or greater sequence identity compared to SEQ IDNO: 27. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-45-1 polypeptide having at least 99.8% or greatersequence identity compared to SEQ ID NO: 29. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-45-1 polypeptidehaving at least 92.3% or greater sequence identity compared to SEQ IDNO: 31. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-45-1 polypeptide having at least 91.1% or greatersequence identity compared to SEQ ID NO: 33. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-45-1 polypeptidehaving at least 95.4% or greater sequence identity compared to SEQ IDNO: 35. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-45-1 polypeptide having at least 93% or greater sequenceidentity compared to SEQ ID NO: 39. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-45-1 polypeptidehaving at least 97.5% or greater sequence identity compared to SEQ IDNO: 43. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-45-1 polypeptide having at least 70% or greater sequenceidentity compared to SEQ ID NO: 45.

Transgenic plants or plant cells comprising a polynucleotide encoding aPIP-45-2 polypeptide are also encompassed by the disclosure. In someembodiments the DNA construct comprises the polynucleotide of SEQ ID NO:109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO:127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 219, SEQ ID NO:221 or SEQ ID NO: 223, that encode the PIP-45-2 polypeptides of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30,SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO:40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ IDNO: 50, SEQ ID NO: 52, SEQ ID NO: 233, SEQ ID NO: 235 and SEQ ID NO:237, respectively. In some embodiments the DNA construct comprises thepolynucleotide of SEQ ID NO: 109, SEQ ID NO: 125, SEQ ID NO: 127, SEQ IDNO: 129, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139,SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ IDNO: 153, SEQ ID NO: 221 or SEQ ID NO: 223 that encode the PIP-45-2polypeptide of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO:22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ IDNO: 34, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQID NO: 235 and SEQ ID NO: 237, respectively. In some embodiments the DNAconstruct comprises a non-genomic nucleic acid molecule encoding thePIP-45-2 polypeptide. In some embodiments the DNA construct comprises apolynucleotide encoding a PIP-45-2 polypeptide sufficiently homologousto the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ IDNO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO:233, SEQ ID NO: 235 and SEQ ID NO: 237 and which has insecticidalactivity. In some embodiments the DNA construct comprises apolynucleotide encoding a PIP-45-2 polypeptide sufficiently homologousto the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ IDNO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQID NO: 46, SEQ ID NO: 235 or SEQ ID NO: 237 and which has insecticidalactivity. “Sufficiently homologous” is used herein to refer to an aminoacid sequence that has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence homology compared to a reference sequence using one ofthe alignment programs described herein using standard parameters. Oneof skill in the art will recognize that these values can beappropriately adjusted to determine corresponding homology of proteinstaking into account amino acid similarity and the like. In someembodiments the sequence homology is against the full length sequence ofa PIP-45-2 polypeptide.

In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-45-2 polypeptide having at least about 50%, 55%, 60%,65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or greater sequence identity compared to SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ IDNO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQID NO: 52, SEQ ID NO: 233, SEQ ID NO: 235 or SEQ ID NO: 237 and whichhas insecticidal activity. In some embodiments the DNA constructcomprises a polynucleotide encoding a PIP-45-2 polypeptide having atleast about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94r/0, 95%, 96%, 97%, 98%, 99% or greater sequence identitycompared to SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO:34, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ IDNO: 235 or SEQ ID NO: 237 and which has insecticidal activity.

In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-45-2 polypeptide having at least 99.2% or greatersequence identity compared to SEQ ID NO: 2. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-45-2 polypeptidehaving at least 98.5% or greater sequence identity compared to SEQ IDNO: 18. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-45-2 polypeptide having at least 96% or greater sequenceidentity compared to SEQ ID NO: 20. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-45-2 polypeptidehaving at least 80% or greater sequence identity compared to SEQ ID NO:22. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-45-2 polypeptide having at least 81% or greater sequenceidentity compared to SEQ ID NO: 24. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-45-2 polypeptidehaving at least 99.5% or greater sequence identity compared to SEQ IDNO: 28. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-45-2 polypeptide having at least 98.5% or greatersequence identity compared to SEQ ID NO: 30. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-45-2 polypeptidehaving at least 92% or greater sequence identity compared to SEQ ID NO:32. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-45-2 polypeptide having at least 91.5% or greatersequence identity compared to SEQ ID NO: 34. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-45-2 polypeptidehaving at least 70% or greater sequence identity compared to SEQ ID NO:36. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-45-2 polypeptide having at least 90% or greater sequenceidentity compared to SEQ ID NO: 40. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-45-2 polypeptidehaving at least 94% or greater sequence identity compared to SEQ ID NO:44. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-45-2 polypeptide having at least 70% or greater sequenceidentity compared to SEQ ID NO: 46.

Transgenic plants or plant cells comprising a polynucleotide encoding aPIP-64-1 polypeptide are also encompassed by the disclosure. In someembodiments the DNA construct comprises the polynucleotide of SEQ ID NO:160, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQID NO: 171, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:178 or SEQ ID NO: 224 that encodes the PIP-64-1 polypeptide of SEQ IDNO: 53, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71and SEQ ID NO: 238, respectively. In some embodiments the DNA constructcomprises the polynucleotide of SEQ ID NO: 160, SEQ ID NO: 165 or SEQ IDNO: 224 that encode the PIP-64-1 polypeptide of SEQ ID NO: 53, SEQ IDNO: 58 and SEQ ID NO: 238. In some embodiments the DNA constructcomprises a non-genomic nucleic acid molecule encoding the PIP-64-1polypeptide. In some embodiments the DNA construct comprises apolynucleotide encoding a PIP-64-1 polypeptide sufficiently homologousto the amino acid sequence of SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO:58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ IDNO: 67, SEQ ID NO: 69, SEQ ID NO: 71 or SEQ ID NO: 238 and which hasinsecticidal activity. In some embodiments the DNA construct comprises apolynucleotide encoding a PIP-64-1 polypeptide sufficiently homologousto the amino acid sequence of SEQ ID NO: 53, SEQ ID NO: 58 or SEQ ID NO:238 and which has insecticidal activity. “Sufficiently homologous” isused herein to refer to an amino acid sequence that has at least about50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology compared to areference sequence using one of the alignment programs described hereinusing standard parameters. One of skill in the art will recognize thatthese values can be appropriately adjusted to determine correspondinghomology of proteins taking into account amino acid similarity and thelike. In some embodiments the sequence homology is against the fulllength sequence of a PIP-64-1 polypeptide. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-64-1 polypeptidehaving at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequenceidentity compared to SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 58, SEQ IDNO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67, SEQID NO: 69, SEQ ID NO: 71 or SEQ ID NO: 238. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-64-1 polypeptidehaving at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequenceidentity compared to SEQ ID NO: 53, SEQ ID NO: 58 or SEQ ID NO: 238.

In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-64-1 polypeptide having at least 75% or greater sequenceidentity compared to SEQ ID NO: 53. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-64-1 polypeptidehaving at least 99.7% or greater sequence identity compared to SEQ IDNO: 58. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-64-1 polypeptide having at least 75% or greater sequenceidentity compared to SEQ ID NO: 238.

Transgenic plants or plant cells comprising a polynucleotide encoding aPIP-64-2 polypeptide are also encompassed by the disclosure. In someembodiments the DNA construct comprises the polynucleotide of SEQ ID NO:161, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQID NO: 170, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO:179 or SEQ ID NO: 225 that encode the PIP-64-2 polypeptide of SEQ ID NO:54, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ IDNO: 63, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72 andSEQ ID NO: 239, respectively. In some embodiments the DNA constructcomprises the polynucleotide of SEQ ID NO: 161, SEQ ID NO: 162, SEQ IDNO: 166 or SEQ ID NO: 225 that encode the PIP-64-2 polypeptide of SEQ IDNO: 54, SEQ ID NO: 55, SEQ ID NO: 59 and SEQ ID NO: 239, respectively.In some embodiments the DNA construct comprises a non-genomic nucleicacid molecule encoding the PIP-64-2 polypeptide. In some embodiments theDNA construct comprises a polynucleotide encoding a PIP-64-2 polypeptidesufficiently homologous to the amino acid sequence of SEQ ID NO: 54, SEQID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63,SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72 or SEQ ID NO:239 and which has insecticidal activity. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-64-2 polypeptidesufficiently homologous to the amino acid sequence of SEQ ID NO: 54, SEQID NO: 55, SEQ ID NO: 59 or SEQ ID NO: 239 and which has insecticidalactivity. “Sufficiently homologous” is used herein to refer to an aminoacid sequence that has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence homology compared to a reference sequence using one ofthe alignment programs described herein using standard parameters. Oneof skill in the art will recognize that these values can beappropriately adjusted to determine corresponding homology of proteinstaking into account amino acid similarity and the like. In someembodiments the sequence homology is against the full length sequence ofa PIP-64-2 polypeptide. In some embodiments the DNA construct comprisesa polynucleotide encoding a PIP-64-2 polypeptide having at least about50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared toSEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO:61, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ IDNO: 72 or SEQ ID NO: 239 and which has insecticidal activity. In someembodiments the DNA construct comprises a polynucleotide encoding aPIP-64-2 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to SEQ ID NO: 54, SEQ ID NO: 55, SEQID NO: 59 or SEQ ID NO: 239 and which has insecticidal activity.

In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-64-2 polypeptide having at least 70% or greater sequenceidentity compared to SEQ ID NO: 54. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-64-2 polypeptidehaving at least 70% or greater sequence identity compared to SEQ ID NO:55. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-64-2 polypeptide having at least 91% or greater sequenceidentity compared to SEQ ID NO: 59. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-64-2 polypeptidehaving at least 70% or greater sequence identity compared to SEQ ID NO:239.

Transgenic plants or plant cells comprising a polynucleotide encoding aPIP-74-1 polypeptide are also encompassed by the disclosure. In someembodiments the DNA construct comprises the polynucleotide of SEQ ID NO:180, SEQ ID NO: 182 or SEQ ID NO: 184 that encode the PIP-74-1polypeptide of SEQ ID NO: 73, SEQ ID NO: 75 and SEQ ID NO: 77,respectively. In some embodiments the DNA construct comprises anon-genomic nucleic acid molecule encoding the PIP-74-1 polypeptide. Insome embodiments the DNA construct comprises a polynucleotide encoding aPIP-74-1 polypeptide sufficiently homologous to the amino acid sequenceof SEQ ID NO: 73, SEQ ID NO: 75 or SEQ ID NO: 77 and which hasinsecticidal activity. “Sufficiently homologous” is used herein to referto an amino acid sequence that has at least about 50%, 55%, 60%, 65%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater sequence homology compared to a reference sequenceusing one of the alignment programs described herein using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding homology ofproteins taking into account amino acid similarity and the like. In someembodiments the sequence homology is against the full length sequence ofa PIP-74-1 polypeptide. In some embodiments the polynucleotide encodes aPIP-74-1 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to SEQ ID NO: 73, SEQ ID NO: 75 orSEQ ID NO: 77.

In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-74-1 polypeptide having at least 75% or greater sequenceidentity compared to SEQ ID NO: 73. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-74-1 polypeptidehaving at least 75% or greater sequence identity compared to SEQ ID NO:75. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-74-1 polypeptide having at least 75% or greater sequenceidentity compared to SEQ ID NO: 77.

Transgenic plants or plant cells comprising a polynucleotide encoding aPIP-74-2 polypeptide are also encompassed by the disclosure. In someembodiments the DNA construct comprises the polynucleotide of SEQ ID NO:181, SEQ ID NO: 183, SEQ ID NO: 185 that encode the PIP-74-2 polypeptideof SEQ ID NO: 74, SEQ ID NO: 76 and SEQ ID NO: 78, respectively. In someembodiments the DNA construct comprises a non-genomic nucleic acidmolecule encoding the PIP-74-2 polypeptide. In some embodiments thepolynucleotide encodes a PIP-74-2 polypeptide sufficiently homologous tothe amino acid sequence of SEQ ID NO: 74, SEQ ID NO: 76 or SEQ ID NO: 78and which has insecticidal activity. “Sufficiently homologous” is usedherein to refer to an amino acid sequence that has at least about 50%,55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater sequence homology compared to areference sequence using one of the alignment programs described hereinusing standard parameters. One of skill in the art will recognize thatthese values can be appropriately adjusted to determine correspondinghomology of proteins taking into account amino acid similarity and thelike. In some embodiments the sequence homology is against the fulllength sequence of a PIP-74-2 polypeptide. In some embodiments thepolynucleotide encodes a PIP-74-2 polypeptide having at least about 50%,55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater sequence identity compared to SEQ IDNO: 74, SEQ ID NO: 76 or SEQ ID NO: 78.

In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-74-2 polypeptide having at least 75% or greater sequenceidentity compared to SEQ ID NO: 74. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-74-2 polypeptidehaving at least 75% or greater sequence identity compared to SEQ ID NO:76. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-74-2 polypeptide having at least 75% or greater sequenceidentity compared to SEQ ID NO: 78.

Transgenic plants or plant cells comprising a polynucleotide encoding aPIP-75 polypeptide are also encompassed by the disclosure. In someembodiments the DNA construct comprises the polynucleotide of SEQ ID NO:186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193 or SEQ ID NO: 194 that encodethe PIP-75 polypeptide of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81,SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO:86 and SEQ ID NO: 87, respectively. In some embodiments the DNAconstruct comprises the polynucleotide of SEQ ID NO: 186, SEQ ID NO:187, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193 orSEQ ID NO: 194 that encode the PIP-75 polypeptide of SEQ ID NO: 79, SEQID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86and SEQ ID NO: 87, respectively. In some embodiments the DNA constructcomprises a non-genomic nucleic acid molecule encoding the PIP-75polypeptide. In some embodiments the DNA construct comprises apolynucleotide encoding a PIP-75 polypeptide sufficiently homologous tothe amino acid sequence of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81,SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO:86 or SEQ ID NO: 87. In some embodiments the DNA construct comprises apolynucleotide encoding a PIP-75 polypeptide sufficiently homologous tothe amino acid sequence of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81,SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87 and whichhas insecticidal activity. “Sufficiently homologous” is used herein torefer to an amino acid sequence that has at least about 50%, 55%, 60%,65%, 70%, 71%, 72%, 73%, 74 cY0 , 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater sequence homology compared to a referencesequence using one of the alignment programs described herein usingstandard parameters. One of skill in the art will recognize that thesevalues can be appropriately adjusted to determine corresponding homologyof proteins taking into account amino acid similarity and the like. Insome embodiments the sequence homology is against the full lengthsequence of a PIP-75 polypeptide. In some embodiments the polynucleotideencodes a PIP-75 polypeptide having at least about 50%, 55%, 60%, 65%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater sequence identity compared to SEQ ID NO: 79, SEQ IDNO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87 and which has insecticidalactivity. In some embodiments the polynucleotide encodes a PIP-75polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to SEQ ID NO: 79, SEQ ID NO: 80, SEQID NO: 81, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87and which has insecticidal activity.

In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-75 polypeptide having at least 75% or greater sequenceidentity compared to SEQ ID NO: 79. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-75 polypeptidehaving at least 75% or greater sequence identity compared to SEQ ID NO:80. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-75 polypeptide having at least 86% or greater sequenceidentity compared to SEQ ID NO: 81. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-75 polypeptidehaving at least 75% or greater sequence identity compared to SEQ ID NO:84. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-75 polypeptide having at least 75% or greater sequenceidentity compared to SEQ ID NO: 85. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-75 polypeptidehaving at least 75% or greater sequence identity compared to SEQ ID NO:86. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-75 polypeptide having at least 75% or greater sequenceidentity compared to SEQ ID NO: 87.

DNA constructs comprising a polynucleotide encoding a PIP-77 polypeptideare also encompassed by the disclosure. In some embodiments the DNAconstruct comprises the polynucleotide of SEQ ID NO: 195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO: 200, SEQ IDNO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205,SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ IDNO: 210, SEQ ID NO: 211, SEQ ID ID NO: 212, SEQ ID NO: 213, SEQ ID NO:214, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQID NO: 230 or SEQ ID NO: 231 that encodes the PIP-77 polypeptide of SEQID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92,SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO:97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ IDNO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106,SEQ ID NO: 107, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ IDNO: 243, SEQ ID NO: 244 and SEQ ID NO: 245, respectively. In someembodiments the DNA construct comprises the polynucleotide of SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ IDNO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204,SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 227, SEQ ID NO: 228 or SEQ IDNO: 231 that encode the PIP-77 polypeptide of SEQ ID NO: 88, SEQ ID NO:89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ IDNO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQID NO: 241, SEQ ID NO: 242 and SEQ ID NO: 245, respectively. In someembodiments the DNA construct comprises a polynucleotide encoding aPIP-77 polypeptide sufficiently homologous to the amino acid sequence ofSEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO:92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ IDNO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101,SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ IDNO: 106, SEQ ID NO: 107, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243,SEQ ID NO: 244 or SEQ ID NO: 245 and which has insecticidal activity. Insome embodiments the DNA construct comprises a polynucleotide encoding aPIP-77 polypeptide sufficiently homologous to the amino acid sequence ofSEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO:93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ IDNO: 98, SEQ ID NO: 100, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 245and which has insecticidal activity. “Sufficiently homologous” is usedherein to refer to an amino acid sequence that has at least about 50%,55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater sequence homology compared to areference sequence using one of the alignment programs described hereinusing standard parameters. One of skill in the art will recognize thatthese values can be appropriately adjusted to determine correspondinghomology of proteins taking into account amino acid similarity and thelike. In some embodiments the sequence homology is against the fulllength sequence of a PIP-77 polypeptide.

In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-77 polypeptide having at least about 50%, 55%, 60%, 65%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater sequence identity compared to SEQ ID NO: 88, SEQ IDNO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98,SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ IDNO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107,SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244 or SEQ IDNO: 245 and which has insecticidal activity. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-77 polypeptidehaving at least 80% or greater sequence identity compared to SEQ ID NO:88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ IDNO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO:102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQID NO: 107, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO:244 or SEQ ID NO: 245 and which has insecticidal activity. In someembodiments the DNA construct comprises a polynucleotide encoding aPIP-77 polypeptide having at least 90% or greater sequence identitycompared to SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91,SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO:96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ IDNO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105,SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 241, SEQ ID NO: 242, SEQ IDNO: 243, SEQ ID NO: 244 or SEQ ID NO: 245 and which has insecticidalactivity. In some embodiments the DNA construct comprises apolynucleotide encoding a PIP-77 polypeptide having at least 95% orgreater sequence identity compared to SEQ ID NO: 88, SEQ ID NO: 89, SEQID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94,SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO:241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244 or SEQ ID NO: 245and which has insecticidal activity.

In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-77 polypeptide having at least about 50%, 55%, 60%, 65%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater sequence identity compared to SEQ ID NO: 88, SEQ IDNO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100,SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 245 and which hasinsecticidal activity. In some embodiments the DNA construct comprises apolynucleotide encoding a PIP-77 polypeptide having at least 80% orgreater sequence identity compared to SEQ ID NO: 88, SEQ ID NO: 89, SEQID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95,SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO:241, SEQ ID NO: 242 or SEQ ID NO: 245 and which has insecticidalactivity. In some embodiments the DNA construct comprises apolynucleotide encoding a PIP-77 polypeptide having at least 95% orgreater sequence identity compared to SEQ ID NO: 88, SEQ ID NO: 89, SEQID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95,SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO:241, SEQ ID NO: 242 or SEQ ID NO: 245 and which has insecticidalactivity.

In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-77 polypeptide having at least 93% or greater sequenceidentity compared to SEQ ID NO: 88. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-77 polypeptidehaving at least 97% or greater sequence identity compared to SEQ ID NO:89. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-77 polypeptide having at least 99% or greater sequenceidentity compared to SEQ ID NO: 90. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-77 polypeptidehaving at least 97% or greater sequence identity compared to SEQ ID NO:92. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-77 polypeptide having at least 87% or greater sequenceidentity compared to SEQ ID NO: 93. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-77 polypeptidehaving at least 86% or greater sequence identity compared to SEQ ID NO:94. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-77 polypeptide having at least 85% or greater sequenceidentity compared to SEQ ID NO: 95. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-77 polypeptidehaving at least 84% or greater sequence identity compared to SEQ ID NO:96. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-77 polypeptide having at least 85% or greater sequenceidentity compared to SEQ ID NO: 97. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-77 polypeptidehaving at least 83% or greater sequence identity compared to SEQ ID NO:98. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-77 polypeptide having at least 80% or greater sequenceidentity compared to SEQ ID NO: 100. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-77 polypeptidehaving at least 85% or greater sequence identity compared to SEQ ID NO:241. In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-77 polypeptide having at least 83% or greater sequenceidentity compared to SEQ ID NO: 242. In some embodiments the DNAconstruct comprises a polynucleotide encoding a PIP-77 polypeptidehaving at least 96% or greater sequence identity compared to SEQ ID NO:245.

Plant Transformation

The methods of the embodiments involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is as used herein meanspresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the embodiments do not depend on a particularmethod for introducing a polynucleotide or polypeptide into a plant,only that the polynucleotide or polypeptides gains access to theinterior of at least one cell of the plant. Methods for introducingpolynucleotide or polypeptides into plants are known in the artincluding, but not limited to, stable transformation methods, transienttransformation methods, and virus-mediated methods.

“Stable transformation” is as used herein means that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” as used herein means that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant. “Plant” as usedherein refers to whole plants, plant organs (e.g., leaves, stems, roots,etc.), seeds, plant cells, propagules, embryos and progeny of the same.Plant cells can be differentiated or undifferentiated (e.g. callus,suspension culture cells, protoplasts, leaf cells, root cells, phloemcells and pollen).

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway, et al., (1986) Biotechniques 4:320-334), electroporation(Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606),Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and5,981,840), direct gene transfer (Paszkowski, et al., (1984) EMBO J.3:2717-2722) and ballistic particle acceleration (see, for example, U.S.Pat. Nos. 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes, et al.,(1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods,ed. Gamborg and Phillips, (Springer-Verlag, Berlin) and McCabe, et al.,(1988) Biotechnology 6:923-926) and Lecl transformation (WO 00/28058).For potato transformation see, Tu, et al., (1998) Plant MolecularBiology 37:829-838 and Chong, et al., (2000) Transgenic Research9:71-78. Additional transformation procedures can be found inWeissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al.,(1987) Particulate Science and Technology 5:27-37 (onion); Christou, etal., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor.Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563(maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein, etal., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984)Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals);Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349(Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman, et al., (Longman, N.Y.), pp. 197-209(pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 andKaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413(rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maizevia Agrobacterium tumefaciens); all of which are herein incorporated byreference.

In specific embodiments, the sequences of the embodiments can beprovided to a plant using a variety of transient transformation methods.Such transient transformation methods include, but are not limited to,the introduction of the insecticidal polypeptide of the disclosure orvariants and fragments thereof directly into the plant or theintroduction of the insecticidal polypeptide of the disclosuretranscript into the plant. Such methods include, for example,microinjection or particle bombardment. See, for example, Crossway, etal., (1986) Mol Gen. Genet. 202:179-185; Nomura, et al., (1986) PlantSci. 44:53-58; Hepler, et al., (1994) Proc. Natl. Acad. Sci.91:2176-2180 and Hush, et al., (1994) The Journal of Cell Science107:775-784, all of which are herein incorporated by reference.Alternatively, the insecticidal polypeptide of the disclosurepolynucleotide can be transiently transformed into the plant usingtechniques known in the art. Such techniques include viral vector systemand the precipitation of the polynucleotide in a manner that precludessubsequent release of the DNA. Thus, transcription from theparticle-bound DNA can occur, but the frequency with which it isreleased to become integrated into the genome is greatly reduced. Suchmethods include the use of particles coated with polyethylimine (PEI;Sigma #P3143).

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO 1999/25855and WO 1999/25853, all of which are herein incorporated by reference.Briefly, the polynucleotide of the embodiments can be contained intransfer cassette flanked by two non-identical recombination sites. Thetransfer cassette is introduced into a plant have stably incorporatedinto its genome a target site which is flanked by two non-identicalrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The polynucleotide ofinterest is thereby integrated at a specific chromosomal position in theplant genome.

Plant transformation vectors may be comprised of one or more DNA vectorsneeded for achieving plant transformation. For example, it is a commonpractice in the art to utilize plant transformation vectors that arecomprised of more than one contiguous DNA segment. These vectors areoften referred to in the art as “binary vectors”. Binary vectors as wellas vectors with helper plasmids are most often used forAgrobacterium-mediated transformation, where the size and complexity ofDNA segments needed to achieve efficient transformation is quite large,and it is advantageous to separate functions onto separate DNAmolecules. Binary vectors typically contain a plasmid vector thatcontains the cis-acting sequences required for T-DNA transfer (such asleft border and right border), a selectable marker that is engineered tobe capable of expression in a plant cell, and a “gene of interest” (agene engineered to be capable of expression in a plant cell for whichgeneration of transgenic plants is desired). Also present on thisplasmid vector are sequences required for bacterial replication. Thecis-acting sequences are arranged in a fashion to allow efficienttransfer into plant cells and expression therein. For example, theselectable marker gene and the pesticidal gene are located between theleft and right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer, as is understood in the art (Hellens and Mullineaux, (2000)Trends in Plant Science 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used forplant transformation. The second plasmid vector is not necessary fortransforming the plants by other methods such as microprojection,microinjection, electroporation, polyethylene glycol, etc.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g., immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene) to recover thetransformed plant cells from a group of untransformed cell mass.Following integration of heterologous foreign DNA into plant cells, onethen applies a maximum threshold level of appropriate selection in themedium to kill the untransformed cells and separate and proliferate theputatively transformed cells that survive from this selection treatmentby transferring regularly to a fresh medium. By continuous passage andchallenge with appropriate selection, one identifies and proliferatesthe cells that are transformed with the plasmid vector. Molecular andbiochemical methods can then be used to confirm the presence of theintegrated heterologous gene of interest into the genome of thetransgenic plant.

Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent. Theshoots are then transferred to a selective rooting medium for recoveringrooted shoot or plantlet. The transgenic plantlet then grows into amature plant and produces fertile seeds (e.g., Hiei, et al., (1994) ThePlant Journal 6:271-282; Ishida, et al., (1996) Nature Biotechnology14:745-750). Explants are typically transferred to a fresh supply of thesame medium and cultured routinely. A general description of thetechniques and methods for generating transgenic plants are found inAyres and Park, (1994) Critical Reviews in Plant Science 13:219-239 andBommineni and Jauhar, (1997) Maydica 42:107-120. Since the transformedmaterial contains many cells; both transformed and non-transformed cellsare present in any piece of subjected target callus or tissue or groupof cells. The ability to kill non-transformed cells and allowtransformed cells to proliferate results in transformed plant cultures.Often, the ability to remove non-transformed cells is a limitation torapid recovery of transformed plant cells and successful generation oftransgenic plants.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick, et al.,(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive or inducible expression ofthe desired phenotypic characteristic identified. Two or moregenerations may be grown to ensure that expression of the desiredphenotypic characteristic is stably maintained and inherited and thenseeds harvested to ensure that expression of the desired phenotypiccharacteristic has been achieved.

The nucleotide sequences of the embodiments may be provided to the plantby contacting the plant with a virus or viral nucleic acids. Generally,such methods involve incorporating the nucleotide construct of interestwithin a viral DNA or RNA molecule. It is recognized that therecombinant proteins of the embodiments may be initially synthesized aspart of a viral polyprotein, which later may be processed by proteolysisin vivo or in vitro to produce the desired insecticidal polypeptide. Itis also recognized that such a viral polyprotein, comprising at least aportion of the amino acid sequence of an insecticidal polypeptide of thedisclosure of the embodiments, may have the desired pesticidal activity.Such viral polyproteins and the nucleotide sequences that encode forthem are encompassed by the embodiments. Methods for providing plantswith nucleotide constructs and producing the encoded proteins in theplants, which involve viral DNA or RNA molecules are known in the art.See, for example, U.S. Pat. Nos. 5,889,191; 5,889,190; 5,866,785;5,589,367 and 5,316,931; herein incorporated by reference.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab, et al., (1990) Proc. Natl. Acad. Sci. USA87:8526-8530; Svab and Maliga, (1993) Proc. Natl. Acad. Sci. USA90:913-917; Svab and Maliga, (1993) EMBO J. 12:601-606. The methodrelies on particle gun delivery of DNA containing a selectable markerand targeting of the DNA to the plastid genome through homologousrecombination. Additionally, plastid transformation can be accomplishedby transactivation of a silent plastid-borne transgene bytissue-preferred expression of a nuclear-encoded and plastid-directedRNA polymerase. Such a system has been reported in McBride, et al.,(1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

The embodiments further relate to plant-propagating material of atransformed plant of the embodiments including, but not limited to,seeds, tubers, corms, bulbs, leaves and cuttings of roots and shoots.

The embodiments may be used for transformation of any plant species,including, but not limited to, monocots and dicots. Examples of plantsof interest include, but are not limited to, corn (Zea mays), Brassicasp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers that may beemployed in practicing the embodiments include, for example, pines suchas loblolly pine (Pinus taeda), slash pine (Pinus ellioth), ponderosapine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Montereypine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Westernhemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Plants of the embodiments include crop plants (for example, corn,alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut,sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

Turf grasses include, but are not limited to: annual bluegrass (Poaannua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poacompressa); Chewing's fescue (Festuca rubra); colonial bentgrass(Agrostis tenuis); creeping bentgrass (Agrostis palustris); crestedwheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyroncristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poapratensis); orchardgrass (Dactylis glomerata); perennial ryegrass(Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba);rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smoothbromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy(Phleum pratense); velvet bentgrass (Agrostis canina); weepingalkaligrass (Puccinellia distans); western wheatgrass (Agropyronsmithh); Bermuda grass (Cynodon spp.); St. Augustine grass (Stenotaphrumsecundatum); zoysia grass (Zoysia spp.); Bahia grass (Paspalum notatum);carpet grass (Axonopus affinis); centipede grass (Eremochloaophiuroides); kikuyu grass (Pennisetum clandesinum); seashore paspalum(Paspalum vaginatum); blue gramma (Bouteloua gracilis); buffalo grass(Buchloe dactyloids); sideoats gramma (Bouteloua curtipendula).

Plants of interest include grain plants that provide seeds of interest,oil-seed plants, and leguminous plants. Seeds of interest include grainseeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc.Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica,maize, alfalfa, palm, coconut, flax, castor, olive, etc. Leguminousplants include beans and peas. Beans include guar, locust bean,fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, favabean, lentils, chickpea, etc.

Transgenic Plants

Transgenic plants or plant cells comprising a polynucleotide encoding aninsecticidal polypeptide are also encompassed by the disclosure.Transgenic plants or plant cells comprising a polynucleotide encoding aPIP-45-1 polypeptide are encompassed by the disclosure. In someembodiments the transgenic plant or plant cell comprises thepolynucleotide of SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ IDNO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122,SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ IDNO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140,SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ IDNO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158,SEQ ID NO: 218, SEQ ID NO: 220 or SEQ ID NO: 222 that encodes thePIP-45-1 polypeptide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35,SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO:45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 232, SEQ IDNO: 234 and SEQ ID NO: 236, respectively. In some embodiments thetransgenic plant or plant cell comprises the polynucleotide of SEQ IDNO: 108, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130,SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ IDNO: 142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 220or SEQ ID NO: 222, that encodes the PIP-45-1 polypeptide of SEQ ID NO:1, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ IDNO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 234 and SEQ ID NO:236, respectively. In some embodiments the transgenic plant or plantcell comprises a non-genomic nucleic acid molecule encoding the PIP-45-1polypeptide. In some embodiments the transgenic plant or plant cellcomprises a polynucleotide encoding a PIP-45-1 polypeptide sufficientlyhomologous to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ IDNO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQID NO: 232, SEQ ID NO: 234 or SEQ ID NO: 236 and which has insecticidalactivity. In some embodiments the transgenic plant or plant cellcomprises a polynucleotide encoding a PIP-45-1 polypeptide sufficientlyhomologous to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 17,SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ IDNO: 43, SEQ ID NO: 45, SEQ ID NO: 234 or SEQ ID NO: 236 and which hasinsecticidal activity. “Sufficiently homologous” is used herein to referto an amino acid sequence that has at least about 50%, 55%, 60%, 65%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater sequence homology compared to a reference sequenceusing one of the alignment programs described herein using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding homology ofproteins taking into account amino acid similarity and the like. In someembodiments the sequence homology is against the full length sequence ofthe PIP-45-1 polypeptide.

In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-45-1 polypeptide having at least about50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared toSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ IDNO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47,SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 232, SEQ ID NO: 234 or SEQ IDNO: 236 and which has insecticidal activity. In some embodiments thetransgenic plant or plant cell comprises a polynucleotide encoding aPIP-45-1 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to SEQ ID NO: 1, SEQ ID NO: 17, SEQID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29,SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO:43, SEQ ID NO: 45, SEQ ID NO: 234 or SEQ ID NO: 236 and which hasinsecticidal activity.

In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-45-1 polypeptide having at least 99.1% orgreater sequence identity compared to SEQ ID NO: 1. In some embodimentsthe transgenic plant or plant cell comprises a polynucleotide encoding aPIP-45-1 polypeptide having at least 99.4% or greater sequence identitycompared to SEQ ID NO: 17. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-45-1 polypeptidehaving at least 99.6% or greater sequence identity compared to SEQ IDNO: 19. In some embodiments the transgenic plant or plant cell comprisesa polynucleotide encoding a PIP-45-1 polypeptide having at least 87% orgreater sequence identity compared to SEQ ID NO: 21. In some embodimentsthe transgenic plant or plant cell comprises a polynucleotide encoding aPIP-45-1 polypeptide having at least 88% or greater sequence identitycompared to SEQ ID NO: 23. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-45-1 polypeptidehaving at least 99.1% or greater sequence identity compared to SEQ IDNO: 27. In some embodiments the transgenic plant or plant cell comprisesa polynucleotide encoding a PIP-45-1 polypeptide having at least 99.8%or greater sequence identity compared to SEQ ID NO: 29. In someembodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-45-1 polypeptide having at least 92.3% orgreater sequence identity compared to SEQ ID NO: 31. In some embodimentsthe transgenic plant or plant cell comprises a polynucleotide encoding aPIP-45-1 polypeptide having at least 91.1% or greater sequence identitycompared to SEQ ID NO: 33. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-45-1 polypeptidehaving at least 95.4% or greater sequence identity compared to SEQ IDNO: 35. In some embodiments the transgenic plant or plant cell comprisesa polynucleotide encoding a PIP-45-1 polypeptide having at least 95% orgreater sequence identity compared to SEQ ID NO: 39. In some embodimentsthe transgenic plant or plant cell comprises a polynucleotide encoding aPIP-45-1 polypeptide having at least 97.5% or greater sequence identitycompared to SEQ ID NO: 43. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-45-1 polypeptidehaving at least 70% or greater sequence identity compared to SEQ ID NO:45. In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-45-1 polypeptide having at least 94% orgreater sequence identity compared to SEQ ID NO: 234. In someembodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-45-1 polypeptide having at least 96% orgreater sequence identity compared to SEQ ID NO: 236.

Transgenic plants or plant cells comprising a polynucleotide encoding aPIP-45-2 polypeptide are also encompassed by the disclosure. In someembodiments the transgenic plant or plant cell comprises thepolynucleotide of SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ IDNO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123,SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ IDNO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141,SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ IDNO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159,SEQ ID NO: 219, SEQ ID NO: 221 or SEQ ID NO: 223 that encodes thePIP-45-2 polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO:36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ IDNO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 233, SEQID NO: 235 and SEQ ID NO: 237, respectively. In some embodiments thetransgenic plant or plant cell comprises the polynucleotide of SEQ IDNO: 109, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131,SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ IDNO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 221or SEQ ID NO: 223 that encode the PIP-45-2 polypeptide of SEQ ID NO: 2,SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ IDNO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 235 and SEQ ID NO: 237,respectively. In some embodiments the transgenic plant or plant cellcomprises a non-genomic nucleic acid molecule encoding the PIP-45-2polypeptide. In some embodiments the transgenic plant or plant cellcomprises a polynucleotide encoding a PIP-45-2 polypeptide sufficientlyhomologous to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO:34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ IDNO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQID NO: 233, SEQ ID NO: 235 and SEQ ID NO: 237 and which has insecticidalactivity. In some embodiments the transgenic plant or plant cellcomprises a polynucleotide encoding a PIP-45-2 polypeptide sufficientlyhomologous to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 18,SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO:30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 40, SEQ IDNO: 44, SEQ ID NO: 46, SEQ ID NO: 235 or SEQ ID NO: 237 and which hasinsecticidal activity. “Sufficiently homologous” is used herein to referto an amino acid sequence that has at least about 50%, 55%, 60%, 65%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91° A), 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater sequence homology compared to a reference sequenceusing one of the alignment programs described herein using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding homology ofproteins taking into account amino acid similarity and the like. In someembodiments the sequence homology is against the full length sequence ofa PIP-45-2 polypeptide.

In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-45-2 polypeptide having at least about50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared toSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ IDNO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 233, SEQ ID NO: 235 or SEQ IDNO: 237 and which has insecticidal activity. In some embodiments thetransgenic plant or plant cell comprises a polynucleotide encoding aPIP-45-2 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to SEQ ID NO: 2, SEQ ID NO: 18, SEQID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30,SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO:44, SEQ ID NO: 46, SEQ ID NO: 235 or SEQ ID NO: 237 and which hasinsecticidal activity.

In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-45-2 polypeptide having at least 99.2% orgreater sequence identity compared to SEQ ID NO: 2. In some embodimentsthe transgenic plant or plant cell comprises a polynucleotide encoding aPIP-45-2 polypeptide having at least 98.5% or greater sequence identitycompared to SEQ ID NO: 18. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-45-2 polypeptidehaving at least 96% or greater sequence identity compared to SEQ ID NO:20. In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-45-2 polypeptide having at least 80% orgreater sequence identity compared to SEQ ID NO: 22. In some embodimentsthe transgenic plant or plant cell comprises a polynucleotide encoding aPIP-45-2 polypeptide having at least 81% or greater sequence identitycompared to SEQ ID NO: 24. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-45-2 polypeptidehaving at least 99.5% or greater sequence identity compared to SEQ IDNO: 28. In some embodiments the transgenic plant or plant cell comprisesa polynucleotide encoding a PIP-45-2 polypeptide having at least 98.5%or greater sequence identity compared to SEQ ID NO: 30. In someembodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-45-2 polypeptide having at least 92% orgreater sequence identity compared to SEQ ID NO: 32. In some embodimentsthe transgenic plant or plant cell comprises a polynucleotide encoding aPIP-45-2 polypeptide having at least 91.5% or greater sequence identitycompared to SEQ ID NO: 34. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-45-2 polypeptidehaving at least 70% or greater sequence identity compared to SEQ ID NO:36. In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-45-2 polypeptide having at least 90% orgreater sequence identity compared to SEQ ID NO: 40. In some embodimentsthe transgenic plant or plant cell comprises a polynucleotide encoding aPIP-45-2 polypeptide having at least 94% or greater sequence identitycompared to SEQ ID NO: 44. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-45-2 polypeptidehaving at least 70% or greater sequence identity compared to SEQ ID NO:46.

Transgenic plants or plant cells comprising a polynucleotide encoding aPIP-64-1 polypeptide are also encompassed by the disclosure. In someembodiments the transgenic plant or plant cell comprises thepolynucleotide of SEQ ID NO: 160, SEQ ID NO: 163, SEQ ID NO: 165, SEQ IDNO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 174,SEQ ID NO: 176, SEQ ID NO: 178 or SEQ ID NO: 224 that encodes thePIP-64-1 polypeptide of SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 58, SEQID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67,SEQ ID NO: 69, SEQ ID NO: 71 and SEQ ID NO: 238, respectively. In someembodiments the transgenic plant or plant cell comprises thepolynucleotide of SEQ ID NO: 160, SEQ ID NO: 165 or SEQ ID NO: 224 thatencode the PIP-64-1 polypeptide of SEQ ID NO: 53, SEQ ID NO: 58 and SEQID NO: 238. In some embodiments the transgenic plant or plant cellcomprises a non-genomic nucleic acid molecule encoding the PIP-64-1polypeptide. In some embodiments the transgenic plant or plant cellcomprises a polynucleotide encoding a PIP-64-1 polypeptide sufficientlyhomologous to the amino acid sequence of SEQ ID NO: 53, SEQ ID NO: 56,SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 or SEQ ID NO: 238 andwhich has insecticidal activity. In some embodiments the transgenicplant or plant cell comprises a polynucleotide encoding a PIP-64-1polypeptide sufficiently homologous to the amino acid sequence of SEQ IDNO: 53, SEQ ID NO: 58 or SEQ ID NO: 238 and which has insecticidalactivity. “Sufficiently homologous” is used herein to refer to an aminoacid sequence that has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%,73%, 74%, 75%, 76)/0, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence homology compared to a reference sequence using one ofthe alignment programs described herein using standard parameters. Oneof skill in the art will recognize that these values can beappropriately adjusted to determine corresponding homology of proteinstaking into account amino acid similarity and the like. In someembodiments the sequence homology is against the full length sequence ofa PIP-64-1 polypeptide. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-64-1 polypeptidehaving at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequenceidentity compared to SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 58, SEQ IDNO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 67, SEQID NO: 69, SEQ ID NO: 71 or SEQ ID NO: 238. In some embodiments thetransgenic plant or plant cell comprises a polynucleotide encoding aPIP-64-1 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to SEQ ID NO: 53, SEQ ID NO: 58 orSEQ ID NO: 238.

In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-64-1 polypeptide having at least 75% orgreater sequence identity compared to SEQ ID NO: 53. In some embodimentsthe transgenic plant or plant cell comprises a polynucleotide encoding aPIP-64-1 polypeptide having at least 99.7% or greater sequence identitycompared to SEQ ID NO: 58. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-64-1 polypeptidehaving at least 75% or greater sequence identity compared to SEQ ID NO:238.

Transgenic plants or plant cells comprising a polynucleotide encoding aPIP-64-2 polypeptide are also encompassed by the disclosure. In someembodiments the transgenic plant or plant cell comprises thepolynucleotide of SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 164, SEQ IDNO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 173, SEQ ID NO: 175,SEQ ID NO: 177, SEQ ID NO: 179 or SEQ ID NO: 225 that encode thePIP-64-2 polypeptide of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 57, SEQID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 68,SEQ ID NO: 70, SEQ ID NO: 72 and SEQ ID NO: 239, respectively. In someembodiments the transgenic plant or plant cell comprises thepolynucleotide of SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 166 or SEQID NO: 225 that encode the PIP-64-2 polypeptide of SEQ ID NO: 54, SEQ IDNO: 55, SEQ ID NO: 59 and SEQ ID NO: 239, respectively. In someembodiments the transgenic plant or plant cell comprises a non-genomicnucleic acid molecule encoding the PIP-64-2 polypeptide. In someembodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-64-2 polypeptide sufficiently homologousto the amino acid sequence SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 57,SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO:68, SEQ ID NO: 70, SEQ ID NO: 72 or SEQ ID NO: 239 and which hasinsecticidal activity. In some embodiments the transgenic plant or plantcell comprises a polynucleotide encoding a PIP-64-2 polypeptidesufficiently homologous to the amino acid sequence of SEQ ID NO: 54, SEQID NO: 55, SEQ ID NO: 59 or SEQ ID NO: 239 and which has insecticidalactivity. “Sufficiently homologous” is used herein to refer to an aminoacid sequence that has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence homology compared to a reference sequence using one ofthe alignment programs described herein using standard parameters. Oneof skill in the art will recognize that these values can beappropriately adjusted to determine corresponding homology of proteinstaking into account amino acid similarity and the like. In someembodiments the sequence homology is against the full length sequence ofa PIP-64-2 polypeptide. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-64-2 polypeptidehaving at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91 ok, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequenceidentity compared to SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 57, SEQ IDNO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 68, SEQID NO: 70, SEQ ID NO: 72 or SEQ ID NO: 239 and which has insecticidalactivity. In some embodiments the transgenic plant or plant cellcomprises a polynucleotide encoding a PIP-64-2 polypeptide having atleast about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identitycompared to SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 59 or SEQ ID NO:239 and which has insecticidal activity.

In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-64-2 polypeptide having at least 70% orgreater sequence identity compared to SEQ ID NO: 54. In some embodimentsthe transgenic plant or plant cell comprises a polynucleotide encoding aPIP-64-2 polypeptide having at least 70% or greater sequence identitycompared to SEQ ID NO: 55. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-64-2 polypeptidehaving at least 91% or greater sequence identity compared to SEQ ID NO:59. In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-64-2 polypeptide having at least 70% orgreater sequence identity compared to SEQ ID NO: 239.

Transgenic plants or plant cells comprising a polynucleotide encoding aPIP-74-1 polypeptide are also encompassed by the disclosure. In someembodiments the transgenic plant or plant cell comprises thepolynucleotide of SEQ ID NO: 180, SEQ ID NO: 182 or SEQ ID NO: 184 thatencode the PIP-74-1 polypeptide of SEQ ID NO: 73, SEQ ID NO: 75 and SEQID NO: 77, respectively. In some embodiments the transgenic plant orplant cell comprises a non-genomic nucleic acid molecule encoding thePIP-74-1 polypeptide. In some embodiments the transgenic plant or plantcell comprises a polynucleotide encoding a PIP-74-1 polypeptidesufficiently homologous to the amino acid sequence of SEQ ID NO: 73, SEQID NO: 75 or SEQ ID NO: 77 and which has insecticidal activity.“Sufficiently homologous” is used herein to refer to an amino acidsequence that has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greatersequence homology compared to a reference sequence using one of thealignment programs described herein using standard parameters. One ofskill in the art will recognize that these values can be appropriatelyadjusted to determine corresponding homology of proteins taking intoaccount amino acid similarity and the like. In some embodiments thesequence homology is against the full length sequence of a PIP-74-1polypeptide. In some embodiments the polynucleotide encodes a PIP-74-1polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to SEQ ID NO: 73, SEQ ID NO: 75 orSEQ ID NO: 77.

In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-74-1 polypeptide having at least 75% orgreater sequence identity compared to SEQ ID NO: 73. In some embodimentsthe transgenic plant or plant cell comprises a polynucleotide encoding aPIP-74-1 polypeptide having at least 75% or greater sequence identitycompared to SEQ ID NO: 75. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-74-1 polypeptidehaving at least 75% or greater sequence identity compared to SEQ ID NO:77.

Transgenic plants or plant cells comprising a polynucleotide encoding aPIP-74-2 polypeptide are also encompassed by the disclosure. In someembodiments the transgenic plant or plant cell comprises thepolynucleotide of SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185 thatencode the PIP-74-2 polypeptide of SEQ ID NO: 74, SEQ ID NO: 76 and SEQID NO: 78, respectively. In some embodiments the transgenic plant orplant cell comprises a non-genomic nucleic acid molecule encoding thePIP-74-2 polypeptide. In some embodiments the polynucleotide encodes aPIP-74-2 polypeptide sufficiently homologous to the amino acid sequenceof SEQ ID NO: 74, SEQ ID NO: 76 or SEQ ID NO: 78 and which hasinsecticidal activity. “Sufficiently homologous” is used herein to referto an amino acid sequence that has at least about 50%, 55%, 60%, 65%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater sequence homology compared to a reference sequenceusing one of the alignment programs described herein using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding homology ofproteins taking into account amino acid similarity and the like. In someembodiments the sequence homology is against the full length sequence ofa PIP-74-2 polypeptide. In some embodiments the polynucleotide encodes aPIP-74-2 polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to SEQ ID NO: 74, SEQ ID NO: 76 orSEQ ID NO: 78.

In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-74-2 polypeptide having at least 75% orgreater sequence identity compared to SEQ ID NO: 74. In some embodimentsthe transgenic plant or plant cell comprises a polynucleotide encoding aPIP-74-2 polypeptide having at least 75% or greater sequence identitycompared to SEQ ID NO: 76. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-74-2 polypeptidehaving at least 75% or greater sequence identity compared to SEQ ID NO:78.

Transgenic plants or plant cells comprising a polynucleotide encoding aPIP-75 polypeptide are also encompassed by the disclosure. In someembodiments the transgenic plant or plant cell comprises thepolynucleotide of SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ IDNO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193or SEQ ID NO: 194 that encode the PIP-75 polypeptide of SEQ ID NO: 79,SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO:84, SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87, respectively. Insome embodiments the transgenic plant or plant cell comprises thepolynucleotide of SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ IDNO: 191, SEQ ID NO: 192, SEQ ID NO: 193 or SEQ ID NO: 194 that encodethe PIP-75 polypeptide of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81,SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 and SEQ ID NO: 87,respectively. In some embodiments the transgenic plant or plant cellcomprises a non-genomic nucleic acid molecule encoding the PIP-75polypeptide. In some embodiments the transgenic plant or plant cellcomprises a polynucleotide encoding a PIP-75 polypeptide sufficientlyhomologous to the amino acid sequence of SEQ ID NO: 79, SEQ ID NO: 80,SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO:85, SEQ ID NO: 86 or SEQ ID NO: 87. In some embodiments the transgenicplant or plant cell comprises a polynucleotide encoding a PIP-75polypeptide sufficiently homologous to the amino acid sequence of SEQ IDNO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 85, SEQID NO: 86 or SEQ ID NO: 87 and which has insecticidal activity.“Sufficiently homologous” is used herein to refer to an amino acidsequence that has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greatersequence homology compared to a reference sequence using one of thealignment programs described herein using standard parameters. One ofskill in the art will recognize that these values can be appropriatelyadjusted to determine corresponding homology of proteins taking intoaccount amino acid similarity and the like. In some embodiments thesequence homology is against the full length sequence of a PIP-75polypeptide. In some embodiments the polynucleotide encodes a PIP-75polypeptide having at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to SEQ ID NO: 79, SEQ ID NO: 80, SEQID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85,SEQ ID NO: 86 or SEQ ID NO: 87 and which has insecticidal activity. Insome embodiments the polynucleotide encodes a PIP-75 polypeptide havingat least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identitycompared to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 84,SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87 and which has insecticidalactivity.

In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-75 polypeptide having at least 75% orgreater sequence identity compared to SEQ ID NO: 79. In some embodimentsthe transgenic plant or plant cell comprises a polynucleotide encoding aPIP-75 polypeptide having at least 75% or greater sequence identitycompared to SEQ ID NO: 80. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-75 polypeptidehaving at least 86% or greater sequence identity compared to SEQ ID NO:81. In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-75 polypeptide having at least 75% orgreater sequence identity compared to SEQ ID NO: 84. In some embodimentsthe transgenic plant or plant cell comprises a polynucleotide encoding aPIP-75 polypeptide having at least 75% or greater sequence identitycompared to SEQ ID NO: 85. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-75 polypeptidehaving at least 75% or greater sequence identity compared to SEQ ID NO:86. In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-75 polypeptide having at least 75% orgreater sequence identity compared to SEQ ID NO: 87.

Transgenic plants or plant cells comprising a polynucleotide encoding aPIP-77 polypeptide are also encompassed by the disclosure. In someembodiments the transgenic plant or plant cell comprises thepolynucleotide of SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO:197, SEQ IDNO:198, SEQ ID NO:199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202,SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ IDNO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211,SEQ ID ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 226, SEQID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230 or SEQ ID NO:231 that encodes the PIP-77 polypeptide of SEQ ID NO: 88, SEQ ID NO: 89,SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO:94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ IDNO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103,SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ IDNO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244 and SEQ ID NO:245, respectively. In some embodiments the transgenic plant or plantcell comprises the polynucleotide of SEQ ID NO: 195, SEQ ID NO:196, SEQID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO: 200, SEQ ID NO: 201,SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ IDNO: 207, SEQ ID NO: 227, SEQ ID NO: 228 or SEQ ID NO: 231 that encodethe PIP-77 polypeptide of SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90,SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO:96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 241, SEQ IDNO: 242 and SEQ ID NO: 245, respectively. In some embodiments thetransgenic plant or plant cell comprises a polynucleotide encoding aPIP-77 polypeptide sufficiently homologous to the amino acid sequence ofSEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO:92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ IDNO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101,SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ IDNO: 106, SEQ ID NO: 107, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242,SEQ ID NO: 243, SEQ ID NO: 244 or SEQ ID NO: 245 and which hasinsecticidal activity. In some embodiments the transgenic plant or plantcell comprises a polynucleotide encoding a PIP-77 polypeptidesufficiently homologous to the amino acid sequence of SEQ ID NO: 88, SEQID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94,SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO:100, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 245 and which hasinsecticidal activity. “Sufficiently homologous” is used herein to referto an amino acid sequence that has at least about 50%, 55%, 60%, 65%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or greater sequence homology compared to a reference sequenceusing one of the alignment programs described herein using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding homology ofproteins taking into account amino acid similarity and the like. In someembodiments the sequence homology is against the full length sequence ofa PIP-77 polypeptide.

In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-77 polypeptide having at least about 50%,55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater sequence identity compared to SEQ IDNO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97,SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO:102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQID NO: 107, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO:243, SEQ ID NO: 244 or SEQ ID NO: 245 and which has insecticidalactivity. In some embodiments the transgenic plant or plant cellcomprises a polynucleotide encoding a PIP-77 polypeptide having at leastabout 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity comparedto SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 92, SEQ IDNO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQID NO: 98, SEQ ID NO: 100, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO:245 and which has insecticidal activity.

In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-77 polypeptide having at least 86% orgreater sequence identity compared to SEQ ID NO: 88. In some embodimentsthe transgenic plant or plant cell comprises a polynucleotide encoding aPIP-77 polypeptide having at least 85% or greater sequence identitycompared to SEQ ID NO: 89. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-77 polypeptidehaving at least 85% or greater sequence identity compared to SEQ ID NO:90. In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-77 polypeptide having at least 85% orgreater sequence identity compared to SEQ ID NO: 91. In some embodimentsthe transgenic plant or plant cell comprises a polynucleotide encoding aPIP-77 polypeptide having at least 85% or greater sequence identitycompared to SEQ ID NO: 92. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-77 polypeptidehaving at least 85% or greater sequence identity compared to SEQ ID NO:93. In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-77 polypeptide having at least 85% orgreater sequence identity compared to SEQ ID NO: 94. In some embodimentsthe transgenic plant or plant cell comprises a polynucleotide encoding aPIP-77 polypeptide having at least 85% or greater sequence identitycompared to SEQ ID NO: 95. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-77 polypeptidehaving at least 80% or greater sequence identity compared to SEQ ID NO:96. In some embodiments the transgenic plant or plant cell comprises apolynucleotide encoding a PIP-77 polypeptide having at least 80% orgreater sequence identity compared to SEQ ID NO: 97. In some embodimentsthe transgenic plant or plant cell comprises a polynucleotide encoding aPIP-77 polypeptide having at least 80% or greater sequence identitycompared to SEQ ID NO: 98. In some embodiments the transgenic plant orplant cell comprises a polynucleotide encoding a PIP-77 polypeptidehaving at least 80% or greater sequence identity compared to SEQ ID NO:100.

Evaluation of Plant Transformation

Following introduction of heterologous foreign DNA into plant cells, thetransformation or integration of heterologous gene in the plant genomeis confirmed by various methods such as analysis of nucleic acids,proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method to screen transformed cells, tissue orshoots for the presence of incorporated gene at the earlier stage beforetransplanting into the soil (Sambrook and Russell, (2001) MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). PCR is carried out using oligonucleotide primersspecific to the gene of interest or Agrobacterium vector background,etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell, (2001) supra). In general, total DNAis extracted from the transformant, digested with appropriaterestriction enzymes, fractionated in an agarose gel and transferred to anitrocellulose or nylon membrane. The membrane or “blot” is then probedwith, for example, radiolabeled 32P target DNA fragment to confirm theintegration of introduced gene into the plant genome according tostandard techniques (Sambrook and Russell, (2001) supra).

In Northern blot analysis, RNA is isolated from specific tissues oftransformant, fractionated in a formaldehyde agarose gel, and blottedonto a nylon filter according to standard procedures that are routinelyused in the art (Sambrook and Russell, (2001) supra). Expression of RNAencoded by the pesticidal gene is then tested by hybridizing the filterto a radioactive probe derived from a pesticidal gene, by methods knownin the art (Sambrook and Russell, (2001) supra).

Western blot, biochemical assays and the like may be carried out on thetransgenic plants to confirm the presence of protein encoded by thepesticidal gene by standard procedures (Sambrook and Russell, 2001,supra) using antibodies that bind to one or more epitopes present on theinsecticidal polypeptide of the disclosure.

Stacking of Traits in Transgenic Plant

Transgenic plants may comprise a stack of one or more insecticidalpolynucleotides disclosed herein with one or more additionalpolynucleotides resulting in the production or suppression of multiplepolypeptide sequences. Transgenic plants comprising stacks ofpolynucleotide sequences can be obtained by either or both oftraditional breeding methods or through genetic engineering methods.These methods include, but are not limited to, breeding individual lineseach comprising a polynucleotide of interest, transforming a transgenicplant comprising a gene disclosed herein with a subsequent gene andco-transformation of genes into a single plant cell. As used herein, theterm “stacked” includes having the multiple traits present in the sameplant (i.e., both traits are incorporated into the nuclear genome, onetrait is incorporated into the nuclear genome and one trait isincorporated into the genome of a plastid or both traits areincorporated into the genome of a plastid). In one non-limiting example,“stacked traits” comprise a molecular stack where the sequences arephysically adjacent to each other. A trait, as used herein, refers tothe phenotype derived from a particular sequence or groups of sequences.Co-transformation of genes can be carried out using singletransformation vectors comprising multiple genes or genes carriedseparately on multiple vectors. If the sequences are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. The traits can beintroduced simultaneously in a co-transformation protocol with thepolynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO1999/25855 and WO 1999/25853, all of which are herein incorporated byreference.

In some embodiments the polynucleotides encoding the insecticidalpolypeptides disclosed herein, alone or stacked with one or moreadditional insect resistance traits can be stacked with one or moreadditional input traits (e.g., herbicide resistance, fungal resistance,virus resistance, stress tolerance, disease resistance, male sterility,stalk strength, and the like) or output traits (e.g., increased yield,modified starches, improved oil profile, balanced amino acids, highlysine or methionine, increased digestibility, improved fiber quality,drought resistance, and the like). Thus, the polynucleotide embodimentscan be used to provide a complete agronomic package of improved cropquality with the ability to flexibly and cost effectively control anynumber of agronomic pests.

Transgenes useful for stacking include but are not limited to:

1. Transgenes that Confer Resistance to Insects or Disease and thatEncode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example, Jones, et al., (1994) Science266:789 (cloning of the tomato Cf-9 gene for resistance to Cladosporiumfulvum); Martin, et al., (1993) Science 262:1432 (tomato Pto gene forresistance to Pseudomonas syringae pv. tomato encodes a protein kinase);Mindrinos, et al., (1994) Cell 78:1089 (Arabidopsis RSP2 gene forresistance to Pseudomonas syringae), McDowell and Woffenden, (2003)Trends Biotechnol. 21(4):178-83 and Toyoda, et al., (2002) TransgenicRes. 11(6):567-82. A plant resistant to a disease is one that is moreresistant to a pathogen as compared to the wild type plant.

(B) Genes encoding a Bacillus thuringiensis protein, a derivativethereof or a synthetic polypeptide modeled thereon. See, for example,Geiser, et al., (1986) Gene 48:109, who disclose the cloning andnucleotide sequence of a Bt delta-endotoxin gene. Moreover, DNAmolecules encoding delta-endotoxin genes can be purchased from AmericanType Culture Collection (Rockville, Md.), for example, under ATCC®Accession Numbers 40098, 67136, 31995 and 31998. Other non-limitingexamples of Bacillus thuringiensis transgenes being geneticallyengineered are given in the following patents and patent applicationsand hereby are incorporated by reference for this purpose: U.S. Pat.Nos. 5,188,960; 5,689,052; 5,880,275; 5,986,177; 6,023,013, 6,060,594,6,063,597, 6,077,824, 6,620,988, 6,642,030, 6,713,259, 6,893,826,7,105,332; 7,179,965, 7,208,474; 7,227,056, 7,288,643, 7,323,556,7,329,736, 7,449,552, 7,468,278, 7,510,878, 7,521,235, 7,544,862,7,605,304, 7,696,412, 7,629,504, 7,705,216, 7,772,465, 7,790,846,7,858,849 and WO 1991/14778; WO 1999/31248; WO 2001/12731; WO 1999/24581and WO 1997/40162.

Genes encoding pesticidal proteins may also be stacked including but arenot limited to: insecticidal proteins from Pseudomonas sp. such asPSEEN3174 (Monalysin, (2011) PLoS Pathogens, 7:1-13), from Pseudomonasprotegens strain CHA0 and Pf-5 (previously fluorescens) (Pechy-Tarr,(2008) Environmental Microbiology 10:2368-2386: GenBank Accession No.EU400157); from Pseudomonas taiwanensis (Liu, et al., (2010) J. Agric.Food Chem. 58:12343-12349) and from Pseudomonas pseudoalcligenes (Zhang,et al., (2009) Annals of Microbiology 59:45-50 and Li, et al., (2007)Plant Cell Tiss. Organ Cult. 89:159-168); insecticidal proteins fromPhotorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) TheOpen Toxinology Journal 3:101-118 and Morgan, et al., (2001) Applied andEnvir. Micro. 67:2062-2069), U.S. Pat. Nos. 6,048,838, and 6,379,946; aPIP-1 polypeptide of US Patent Publication Number US2014-0007297-A1; anAflP-1A and/or AflP-1B polypeptides of US Patent Publication NumberUS2014-0033361; a PHI-4 polypeptides of U.S. Ser. No. 13/839,702; PIP-47polypeptides of of PCT Serial Number PCT/US14/51063; a PHI-4 polypeptideof US patent Publication US20140274885 or PCT Patent PublicationWO2014/150914; a PIP-72 polypeptide of PCT Serial Number PCT/US14/55128;the insecticidal proteins of U.S. Ser. No. 61/863,761 and 61/863,763;and δ-endotoxins including, but not limited to, the Cry1, Cry2, Cry3,Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14,Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21, Cry22, Cry23, Cry24,Cry25, Cry26, Cry27, Cry28, Cry29, Cry30, Cry31, Cry32, Cry33, Cry34,Cry35, Cry36, Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44,Cry45, Cry 46, Cry47, Cry49, Cry 51, Cry52, Cry 53, Cry 54, Cry55,Cry56, Cry57, Cry58, Cry59. Cry60, Cry61, Cry62, Cry63, Cry64, Cry65,Cry66, Cry67, Cry68, Cry69, Cry70, Cry71 and Cry72 classes ofδ-endotoxin genes and the B. thuringiensis cytolytic Cyt1 and Cyt2genes. Members of these classes of B. thuringiensis insecticidalproteins include, but are not limited to Cry1Aa1 (Accession #AAA22353);Cry1Aa2 (Accession #Accession #AAA22552); Cry1Aa3 (Accession #BAA00257);Cry1Aa4 (Accession #CAA31886); Cry1Aa5 (Accession #BAAO4468); Cry1Aa6(Accession #AAA86265); Cry1Aa7 (Accession #AAD46139); Cry1Aa8 (Accession#I26149); Cry1Aa9 (Accession #BAA77213); Cry1Aa10 (Accession #AAD55382);Cry1Aa11 (Accession #CAA70856); Cry1Aa12 (Accession #AAP80146); Cry1Aa13(Accession #AAM44305); Cry1Aa14 (Accession #AAP40639); Cry1Aa15(Accession #AAY66993); Cry1Aa16 (Accession #HQ439776); Cry1Aa17(Accession #HQ439788); Cry1Aa18 (Accession #HQ439790); Cry1Aa19(Accession #HQ685121); Cry1Aa20 (Accession #JF340156); Cry1Aa21(Accession #JN651496); Cry1Aa22 (Accession #KC158223); Cry1Ab1(Accession #AAA22330); Cry1Ab2 (Accession #AAA22613); Cry1Ab3 (Accession#AAA22561); Cry1Ab4 (Accession #BAAO0071); Cry1Ab5 (Accession#CAA28405); Cry1Ab6 (Accession #AAA22420); Cry1Ab7 (Accession#CAA31620); Cry1Ab8 (Accession #AAA22551); Cry1Ab9 (Accession#CAA38701); Cry1Ab10 (Accession #A29125); Cry1Ab11 (Accession #I12419);Cry1Ab12 (Accession #AAC64003); Cry1Ab13 (Accession #AAN76494); Cry1Ab14(Accession #AAG16877); Cry1Ab15 (Accession #AAO13302); Cry1Ab16(Accession #AAK55546); Cry1Ab17 (Accession #AAT46415); Cry1Ab18(Accession #AAQ88259); Cry1Ab19 (Accession #AAW31761); Cry1Ab20(Accession #ABB72460); Cry1Ab21 (Accession #ABS18384); Cry1Ab22(Accession #ABW87320); Cry1Ab23 (Accession #HQ439777); Cry1Ab24(Accession #HQ439778); Cry1Ab25 (Accession #HQ685122); Cry1Ab26(Accession #HQ847729); Cry1Ab27 (Accession #JN135249); Cry1Ab28(Accession #JN135250); Cry1Ab29 (Accession #JN135251); Cry1Ab30(Accession #JN135252); Cry1Ab31 (Accession #JN135253); Cry1Ab32(Accession #JN135254); Cry1Ab33 (Accession #AAS93798); Cry1Ab34(Accession #KC156668); Cry1Ab-like (Accession #AAK14336); Cry1Ab-like(Accession #AAK14337); Cry1Ab-like (Accession #AAK14338); Cry1Ab-like(Accession #ABG88858); Cry1Ac1 (Accession #AAA22331); Cry1Ac2 (Accession#AAA22338); Cry1Ac3 (Accession #CAA38098); Cry1Ac4 (Accession#AAA73077); Cry1Ac5 (Accession #AAA22339); Cry1Ac6 (Accession#AAA86266); Cry1Ac7 (Accession #AAB46989); Cry1Ac8 (Accession#AAC44841); Cry1Ac9 (Accession #AAB49768); Cry1Ac10 (Accession#CAA05505); Cry1Ac11 (Accession #CAA10270); Cry1Ac12 (Accession#I12418); Cry1Ac13 (Accession #AAD38701); Cry1Ac14 (Accession#AAQ06607); Cry1Ac15 (Accession #AAN07788); Cry1Ac16 (Accession#AAU87037); Cry1Ac17 (Accession #AAX18704); Cry1Ac18 (Accession#AAY88347); Cry1Ac19 (Accession #ABD37053); Cry1Ac20 (Accession#ABB89046); Cry1Ac21 (Accession #AAY66992); Cry1Ac22 (Accession#ABZ01836); Cry1Ac23 (Accession #CA030431); Cry1Ac24 (Accession#ABL01535); Cry1Ac25 (Accession #FJ513324); Cry1Ac26 (Accession#FJ617446); Cry1Ac27 (Accession #FJ617447); Cry1Ac28 (Accession#ACM90319); Cry1Ac29 (Accession #D0438941); Cry1Ac30 (Accession#GQ227507); Cry1Ac31 (Accession #GU446674); Cry1Ac32 (Accession#HM061081); Cry1Ac33 (Accession #GQ866913); Cry1Ac34 (Accession#HQ230364); Cry1Ac35 (Accession #JF340157); Cry1Ac36 (Accession#JN387137); Cry1Ac37 (Accession #JQ317685); Cry1Ad1 (Accession#AAA22340); Cry1Ad2 (Accession #CAA01880); Cry1AO (Accession #AAA22410);Cry1Af1 (Accession #AAB82749); Cry1Ag 1 (Accession #AAD46137); Cry1Ah1(Accession #AAQ14326); Cry1Ah2 (Accession #ABB76664); Cry1Ah3 (Accession#HQ439779); Cry1Ai1 (Accession #AAO39719); Cry1Ai2 (Accession#HQ439780); Cry1A-like (Accession #AAK14339); Cry1Ba1 (Accession#CAA29898); Cry1Ba2 (Accession #CAA65003); Cry1Ba3 (Accession#AAK63251); Cry1Ba4 (Accession #AAK51084); Cry1Ba5 (Accession#AB020894); Cry1Ba6 (Accession #ABL60921); Cry1Ba1 (Accession#HQ439781); Cry1Bb1 (Accession #AAA22344); Cry1Bb2 (Accession#HQ439782); Cry1Bc1 (Accession #CAA86568); Cry1Bd1 (Accession#AAD10292); Cry1Bd2 (Accession #AAM93496); Cry1Be1 (Accession#AAC32850); Cry1Be2 (Accession #AAQ52387); Cry1Be3 (Accession#ACV96720); Cry1Be4 (Accession #HM070026); Cry1Bf1 (Accession#CAC50778); Cry1Bf2 (Accession #AAQ52380); Cry1Bg 1 (Accession#AAO39720); Cry1Bh1 (Accession #HQ589331); Cry1BO (Accession #KC156700);Cry1Ca1 (Accession #CAA30396); Cry1Ca2 (Accession #CAA31951); Cry1Ca3(Accession #AAA22343); Cry1Ca4 (Accession #CAA01886); Cry1Ca5 (Accession#CAA65457); Cry1Ca6 [1] (Accession #AAF37224); Cry1Ca1 (Accession#AAG50438); Cry1Ca8 (Accession #AAM00264); Cry1Ca9 (Accession#AAL79362); Cry1Cal 0 (Accession #AAN16462); Cry1Cal 1 (Accession#AAX53094); Cry1Cal 2 (Accession #HM070027); Cry1Cal 3 (Accession#HQ412621); Cry1Cal 4 (Accession #JN651493); Cry1Cb1 (Accession#M97880); Cry1Cb2 (Accession #AAG35409); Cry1Cb3 (Accession #ACD50894);Cry1Cb-like (Accession #AAX63901); Cry1Da1 (Accession #CAA38099);Cry1Da2 (Accession #176415); Cry1Da3 (Accession #HQ439784); Cry1Db1(Accession #CAA80234); Cry1Db2 (Accession #AAK48937); Cry1Dc1 (Accession#ABK35074); Cry1Ea1 (Accession #CAA37933); Cry1Ea2 (Accession#CAA39609); Cry1Ea3 (Accession #AAA22345); Cry1Ea4 (Accession#AAD04732); Cry1Ea5 (Accession #A15535); Cry1Ea6 (Accession #AAL50330);Cry1Ea7 (Accession #AAW72936); Cry1Ea8 (Accession #ABX11258); Cry1Ea9(Accession #HQ439785); Cry1Ea10 (Accession #ADR00398); Cry1Ea11(Accession #JQ652456); Cry1Eb1 (Accession #AAA22346); Cry1Fa1 (Accession#AAA22348); Cry1Fa2 (Accession #AAA22347); Cry1Fa3 (Accession#HM070028); Cry1Fa4 (Accession #HM439638); Cry1Fb1 (Accession#CAA80235); Cry1Fb2 (Accession #BAA25298); Cry1Fb3 (Accession#AAF21767); Cry1Fb4 (Accession #AAC10641); Cry1Fb5 (Accession#AAO13295); Cry1Fb6 (Accession #ACD50892); Cry1Fb7 (Accession#ACD50893); Cry1Ga1 (Accession #CAA80233); Cry1Ga2 (Accession#CAA70506); Cry1Gb1 (Accession #AAD10291); Cry1Gb2 (Accession#AAO13756); Cry1Gc1 (Accession #AAQ52381); Cry1Ha1 (Accession#CAA80236); Cry1Hb1 (Accession #AAA79694); Cry1Hb2 (Accession#HQ439786); Cry1H-like (Accession #AAF01213); Cry1Ia1 (Accession#CAA44633); Cry1Ia2 (Accession #AAA22354); Cry11a3 (Accession#AAC36999); Cry1Ia4 (Accession #AAB00958); Cry1Ia5 (Accession#CAA70124); Cry1Ia6 (Accession #AAC26910); Cry11a7 (Accession#AAM73516); Cry1Ia8 (Accession #AAK66742); Cry1Ia9 (Accession#AAQ08616); Cry1Ia10 (Accession #AAP86782); Cry1Ia11 (Accession#CAC85964); Cry1Ia12 (Accession #AAV53390); Cry1Ia13 (Accession#ABF83202); Cry1Ia14 (Accession #ACG63871); Cry1Ia15 (Accession#FJ617445); Cry1Ia16 (Acession #FJ617448); Cry1Ia17 (Accession#GU989199); Cry1Ia18 (Accession #ADK23801); Cry1Ia19 (Accession#HQ439787); Cry11a20 (Accession #JQ228426); Cry11a21 (Accession#JQ228424); Cry1Ia22 (Accession #JQ228427); Cry1Ia23 (Accession#JQ228428); Cry1Ia24 (Accession #JQ228429); Cry1Ia25 (Accession#JQ228430); Cry11a26 (Accession #JQ228431); Cry1Ia27 (Accession#JQ228432); Cry1Ia28 (Accession #JQ228433); Cry1Ia29 (Accession#JQ228434); Cry11a30 (Accession #JQ317686); Cry11a31 (Accession#JX944038); Cry11a32 (Accession #JX944039); Cry11a33 (Accession#JX944040); Cry1Ib1 (Accession #AAA82114); Cry1Ib2 (Accession#ABW88019); Cry1Ib3 (Accession #ACD75515); Cry1Ib4 (Accession#HM051227); Cry1Ib5 (Accession #HM070028); Cry1Ib6 (Accession#ADK38579); Cry1Ib7 (Accession #JN571740); Cry1Ib8 (Accession#JN675714); Cry1Ib9 (Accession #JN675715); Cry1Ib10 (Accession#JN675716); Cry1Ib11 (Accession #JQ228423); Cry1Ic1 (Accession#AAC62933); Cry1Ic2 (Accession #AAE71691); Cry1Id1 (Accession#AAD44366); Cry1Id2 (Accession #JQ228422); Cry1Ie1 (Accession#AAG43526); Cry1Ie2 (Accession #HM439636); Cry1Ie3 (Accession#KC156647); Cry11e4 (Accession #KC156681); Cry11f1 (Accession#AAQ52382); Cry1Ig1 (Accession #KC156701); Cry1I-like (Accession#AAC31094); Cry1I-like (Accession #ABG88859); Cry1Ja1 (Accession#AAA22341); Cry1Ja2 (Accession #HM070030); Cry1Ja3 (Accession#JQ228425); Cry1Jb1 (Accession #AAA98959); Cry1Jc1 (Accession#AAC31092); Cry1Jc2 (Accession #AAQ52372); Cry1Jd1 (Accession#CAC50779); Cry1Ka1 (Accession #AAB00376); Cry1Ka2 (Accession#HQ439783); Cry1La1 (Accession #AAS60191); Cry1La2 (Accession#HM070031); Cry1Ma1 (Accession #FJ884067); Cry1Ma2 (Accession#KC156659); Cry1Na1 (Accession #KC156648); Cry1Nb1 (Accession#KC156678); Cry1-like (Accession #AAC31091); Cry2Aa1 (Accession#AAA22335); Cry2Aa2 (Accession #AAA83516); Cry2Aa3 (Accession #D86064);Cry2Aa4 (Accession #AAC04867); Cry2Aa5 (Accession #CAA10671); Cry2Aa6(Accession #CAA10672); Cry2Aa7 (Accession #CAA10670); Cry2Aa8 (Accession#AAO13734); Cry2Aa9 (Accession #AAO13750); Cry2Aa10 (Accession#AAQ04263); Cry2Aa11 (Accession #AAQ52384); Cry2Aa12 (Accession#AB183671); Cry2Aa13 (Accession #ABL01536); Cry2Aa14 (Accession#ACF04939); Cry2Aa15 (Accession #JN426947); Cry2Ab1 (Accession#AAA22342); Cry2Ab2 (Accession #CAA39075); Cry2Ab3 (Accession#AAG36762); Cry2Ab4 (Accession #AAO13296); Cry2Ab5 (Accession#AAQ04609); Cry2Ab6 (Accession #AAP59457); Cry2Ab7 (Accession#AAZ66347); Cry2Ab8 (Accession #ABC95996); Cry2Ab9 (Accession#ABC74968); Cry2Ab10 (Accession #EF157306); Cry2Ab11 (Accession#CAM84575); Cry2Ab12 (Accession #ABM21764); Cry2Ab13 (Accession#ACG76120); Cry2Ab14 (Accession #ACG76121); Cry2Ab15 (Accession#HM037126); Cry2Ab16 (Accession #GQ866914); Cry2Ab17 (Accession#HQ439789); Cry2Ab18 (Accession #JN135255); Cry2Ab19 (Accession#JN135256); Cry2Ab20 (Accession #JN135257); Cry2Ab21 (Accession#JN135258); Cry2Ab22 (Accession #JN135259); Cry2Ab23 (Accession#JN135260); Cry2Ab24 (Accession #JN135261); Cry2Ab25 (Accession#JN415485); Cry2Ab26 (Accession #JN426946); Cry2Ab27 (Accession#JN415764); Cry2Ab28 (Accession #JN651494); Cry2Ac1 (Accession#CAA40536); Cry2Ac2 (Accession #AAG35410); Cry2Ac3 (Accession#AAQ52385); Cry2Ac4 (Accession #ABC95997); Cry2Ac5 (Accession#ABC74969); Cry2Ac6 (Accession #ABC74793); Cry2Ac7 (Accession#CAL18690); Cry2Ac8 (Accession #CAM09325); Cry2Ac9 (Accession#CAM09326); Cry2Ac10 (Accession #ABN15104); Cry2Ac11 (Accession#CAM83895); Cry2Ac12 (Accession #CAM83896); Cry2Ad1 (Accession#AAF09583); Cry2Ad2 (Accession #ABC86927); Cry2Ad3 (Accession#CAK29504); Cry2Ad4 (Accession #CAM32331); Cry2Ad5 (Accession#CA078739); Cry2Ae1 (Accession #AAQ52362); Cry2Af1 (Accession#AB030519); Cry2Af2 (Accession #GQ866915); Cry2Ag1 (Accession#ACH91610); Cry2Ah1 (Accession #EU939453); Cry2Ah2 (Accession#ACL80665); Cry2Ah3 (Accession #GU073380); Cry2Ah4 (Accession#KC156702); Cry2Ai1 (Accession #FJ788388); Cry2Aj (Accession #); Cry2Ak1(Accession #KC156660); Cry2Ba1 (Accession #KC156658); Cry3Aa1 (Accession#AAA22336); Cry3Aa2 (Accession #AAA22541); Cry3Aa3 (Accession#CAA68482); Cry3Aa4 (Accession #AAA22542); Cry3Aa5 (Accession#AAA50255); Cry3Aa6 (Accession #AAC43266); Cry3Aa7 (Accession#CAB41411); Cry3Aa8 (Accession #AAS79487); Cry3Aa9 (Accession#AAWO5659); Cry3Aa10 (Accession #AAU29411); Cry3Aa11 (Accession#AAW82872); Cry3Aa12 (Accession #ABY49136); Cry3Ba1 (Accession#CAA34983); Cry3Ba2 (Accession #CAA00645); Cry3Ba3 (Accession#JQ397327); Cry3Bb1 (Accession #AAA22334); Cry3Bb2 (Accession#AAA74198); Cry3Bb3 (Accession #I15475); Cry3Ca1 (Accession #CAA42469);Cry4Aa1 (Accession #CAA68485); Cry4Aa2 (Accession #BAAO0179); Cry4Aa3(Accession #CAD30148); Cry4Aa4 (Accession #AFB18317); Cry4A-like(Accession #AAY96321); Cry4Ba1 (Accession #CAA30312); Cry4Ba2 (Accession#CAA30114); Cry4Ba3 (Accession #AAA22337); Cry4Ba4 (Accession#BAAO0178); Cry4Ba5 (Accession #CAD30095); Cry4Ba-like (Accession#ABC47686); Cry4Ca1 (Accession #EU646202); Cry4Cb1 (Accession#FJ403208); Cry4Cb2 (Accession #FJ597622); Cry4Cc1 (Accession#FJ403207); Cry5Aa1 (Accession #AAA67694); Cry5Ab1 (Accession#AAA67693); Cry5Ac1 (Accession #I34543); Cry5Ad1 (Accession #ABQ82087);Cry5Ba1 (Accession #AAA68598); Cry5Ba2 (Accession #ABW88931); Cry5Ba3(Accession #AFJQ4417); Cry5Ca1 (Accession #HM461869); Cry5Ca2 (Accession#ZP 04123426); Cry5Da1 (Accession #HM461870); Cry5Da2 (Accession #ZP04123980); Cry5Ea1 (Accession #HM485580); Cry5Ea2 (Accession #ZP04124038); Cry6Aa1 (Accession #AAA22357); Cry6Aa2 (Accession #AAM46849);Cry6Aa3 (Accession #ABHQ3377); Cry6Ba1 (Accession #AAA22358); Cry7Aa1(Accession #AAA22351); Cry7Ab1 (Accession #AAA21120); Cry7Ab2 (Accession#AAA21121); Cry7Ab3 (Accession #ABX24522); Cry7Ab4 (Accession#EU380678); Cry7Ab5 (Accession #ABX79555); Cry7Ab6 (Accession#AC144005); Cry7Ab7 (Accession #ADB89216); Cry7Ab8 (Accession#GU145299); Cry7Ab9 (Accession #ADD92572); Cry7Ba1 (Accession#ABB70817); Cry7Bb1 (Accession #KC156653); Cry7Ca1 (Accession#ABR67863); Cry7Cb1 (Accession #KC156698); Cry7Da1 (Accession#ACQ99547); Cry7Da2 (Accession #HM572236); Cry7Da3 (Accession#KC156679); Cry7Ea1 (Accession #HM035086); Cry7Ea2 (Accession#HM132124); Cry7Ea3 (Accession #EEM19403); Cry7Fa1 (Accession#HM035088); Cry7Fa2 (Accession #EEM19090); Cry7Fb1 (Accession#HM572235); Cry7Fb2 (Accession #KC156682); Cry7Ga1 (Accession#HM572237); Cry7Ga2 (Accession #KC156669); Cry7Gb1 (Accession#KC156650); Cry7Gc1 (Accession #KC156654); Cry7Gd1 (Accession#KC156697); Cry7Ha1 (Accession #KC156651); Cry71a1 (Accession#KC156665); Cry7Ja1 (Accession #KC156671); Cry7Ka1 (Accession#KC156680); Cry7Kb1 (Accession #BAM99306); Cry7La1 (Accession#BAM99307); Cry8Aa1 (Accession #AAA21117); Cry8Ab1 (Accession#EU044830); Cry8Ac1 (Accession #KC156662); Cry8Ad1 (Accession#KC156684); Cry8Ba1 (Accession #AAA21118); Cry8Bb1 (Accession#CAD57542); Cry8Bc1 (Accession #CAD57543); Cry8Ca1 (Accession#AAA21119); Cry8Ca2 (Accession #AAR98783); Cry8Ca3 (Accession#EU625349); Cry8Ca4 (Accession #ADB54826); Cry8Da1 (Accession#BAC07226); Cry8Da2 (Accession #BD133574); Cry8Da3 (Accession#BD133575); Cry8Db1 (Accession #BAF93483); Cry8Ea1 (Accession#AAQ73470); Cry8Ea2 (Accession #EU047597); Cry8Ea3 (Accession#KC855216); Cry8Fa1 (Accession #AAT48690); Cry8Fa2 (Accession#HQ174208); Cry8Fa3 (Accession #AFH78109); Cry8Ga1 (Accession#AAT46073); Cry8Ga2 (Accession #ABC42043); Cry8Ga3 (Accession#FJ198072); Cry8Ha1 (Accession #AAW81032); Cry8la1 (Accession#EU381044); Cry8Ia2 (Accession #GU073381); Cry8Ia3 (Accession#HM044664); Cry8Ia4 (Accession #KC156674); Cry8Ib1 (Accession#GU325772); Cry8Ib2 (Accession #KC156677); Cry8Ja1 (Accession#EU625348); Cry8Ka1 (Accession #FJ422558); Cry8Ka2 (Accession#ACN87262); Cry8Kb1 (Accession #HM123758); Cry8Kb2 (Accession#KC156675); Cry8La1 (Accession #GU325771); Cry8Ma1 (Accession#HM044665); Cry8Ma2 (Accession #EEM86551); Cry8Ma3 (Accession#HM210574); Cry8Na1 (Accession #HM640939); Cry8Pa1 (Accession#HQ388415); Cry8Qa1 (Accession #HQ441166); Cry8Qa2 (Accession#KC152468); Cry8Ra1 (Accession #AFP87548); Cry8Sa1 (Accession#JQ740599); Cry8Ta1 (Accession #KC156673); Cry8-like (Accession#FJ770571); Cry8-like (Accession #ABS53003); Cry9Aa1 (Accession#CAA41122); Cry9Aa2 (Accession #CAA41425); Cry9Aa3 (Accession#GQ249293); Cry9Aa4 (Accession #GQ249294); Cry9Aa5 (Accession#JX174110); Cry9Aa like (Accession #AAQ52376); Cry9Ba1 (Accession#CAA52927); Cry9Ba2 (Accession #GU299522); Cry9Bb1 (Accession#AAV28716); Cry9Ca1 (Accession #CAA85764); Cry9Ca2 (Accession#AAQ52375); Cry9Da1 (Accession #BAA19948); Cry9Da2 (Accession#AAB97923); Cry9Da3 (Accession #GQ249293); Cry9Da4 (Accession#GQ249297); Cry9Db1 (Accession #AAX78439); Cry9Dc1 (Accession#KC156683); Cry9Ea1 (Accession #BAA34908); Cry9Ea2 (Accession#AAO12908); Cry9Ea3 (Accession #ABM21765); Cry9Ea4 (Accession#ACE88267); Cry9Ea5 (Accession #ACF04743); Cry9Ea6 (Accession#ACG63872); Cry9Ea7 (Accession #FJ380927); Cry9Ea8 (Accession#GQ249292); Cry9Ea9 (Accession #JN651495); Cry9Eb1 (Accession#CAC50780); Cry9Eb2 (Accession #GQ249298); Cry9Eb3 (Accession#KC156646); Cry9Ec1 (Accession #AAC63366); Cry9Ed1 (Accession#AAX78440); Cry9Ee1 (Accession #GQ249296); Cry9Ee2 (Accession#KC156664); Cry9Fa1 (Accession #KC156692); Cry9Ga1 (Accession#KC156699); Cry9-like (Accession #AAC63366); Cry10Aa1 (Accession#AAA22614); Cry10Aa2 (Accession #E00614); Cry10Aa3 (Accession#CAD30098); Cry10Aa4 (Accession #AFB18318); Cry10A-like (Accession#D0167578); Cry11Aa1 (Accession #AAA22352); Cry11Aa2 (Accession#AAA22611); Cry11Aa3 (Accession #CAD30081); Cry11Aa4 (Accession#AFB18319); Cry11Aa-like (Accession #DQ166531); Cry11Ba1 (Accession#CAA60504); Cry11Bb1 (Accession #AAC97162); Cry11Bb2 (Accession#HM068615); Cry12Aa1 (Accession #AAA22355); Cry13Aa1 (Accession#AAA22356); Cry14Aa1 (Accession #AAA21516); Cry14Ab1 (Accession#KC156652); Cry15Aa1 (Accession #AAA22333); Cry16Aal (Accession#CAA63860); Cry17Aa1 (Accession #CAA67841); Cry18Aa1 (Accession#CAA67506); Cry18Ba1 (Accession #AAF89667); Cry18Ca1 (Accession#AAF89668); Cry19Aa1 (Accession #CAA68875); Cry19Ba1 (Accession#BAA32397); Cry19Cal (Accession #AFM37572); Cry20Aa1 (Accession#AAB93476); Cry20Ba1 (Accession #ACS93601); Cry20Ba2 (Accession#KC156694); Cry20-like (Accession #GQ144333); Cry21 Aa1 (Accession#I32932); Cry21 Aa2 (Accession #I66477); Cry21Ba1 (Accession #BAC06484);Cry21Ca1 (Accession #JF521577); Cry21Ca2 (Accession #KC156687); Cry21Da1 (Accession #JF521578); Cry22Aa1 (Accession #I34547); Cry22Aa2(Accession #CAD43579); Cry22Aa3 (Accession #ACD93211); Cry22Ab1(Accession #AAK50456); Cry22Ab2 (Accession #CAD43577); Cry22Ba1(Accession #CAD43578); Cry22Bb1 (Accession #KC156672); Cry23Aa1(Accession #AAF76375); Cry24Aa1 (Accession #AAC61891); Cry24Ba1(Accession #BAD32657); Cry24Ca1 (Accession #CAJ43600); Cry25Aa1(Accession #AAC61892); Cry26Aa1 (Accession #AAD25075); Cry27Aa1(Accession #BAA82796); Cry28Aa1 (Accession #AAD24189); Cry28Aa2(Accession #AAGQ0235); Cry29Aa1 (Accession #CAC80985); Cry30Aa1(Accession #CAC80986); Cry30Ba1 (Accession #BAD00052); Cry30Ca1(Accession #BAD67157); Cry30Ca2 (Accession #ACU24781); Cry30Da1(Accession #EF095955); Cry30Db1 (Accession #BAE80088); Cry30Ea1(Accession #ACC95445); Cry30Ea2 (Accession #FJ499389); Cry30Fa1(Accession #ACI22625); Cry30Ga1 (Accession #ACG60020); Cry30Ga2(Accession #HQ638217); Cry31 Aa1 (Accession #BAB11757); Cry31 Aa2(Accession #AAL87458); Cry31 Aa3 (Accession #BAE79808); Cry31 Aa4(Accession #BAF32571); Cry31 Aa5 (Accession #BAF32572); Cry31 Aa6(Accession #BA144026); Cry31 Abl (Accession #BAE79809); Cry31 Ab2(Accession #BAF32570); Cry31 Ac1 (Accession #BAF34368); Cry31 Ac2(Accession #AB731600); Cry31 Ad1 (Accession #BA144022); Cry32Aa1(Accession #AAG36711); Cry32Aa2 (Accession #GU063849); Cry32Ab1(Accession #GU063850); Cry32Ba1 (Accession #BAB78601); Cry32Ca1(Accession #BAB78602); Cry32Cb1 (Accession #KC156708); Cry32Da1(Accession #BAB78603); Cry32Ea1 (Accession #GU324274); Cry32Ea2(Accession #KC156686); Cry32Eb1 (Accession #KC156663); Cry32Fa1(Accession #KC156656); Cry32Ga1 (Accession #KC156657); Cry32Ha1(Accession #KC156661); Cry32Hb1 (Accession #KC156666); Cry321a1(Accession #KC156667); Cry32Ja1 (Accession #KC156685); Cry32Ka1(Accession #KC156688); Cry32La1 (Accession #KC156689); Cry32Ma1(Accession #KC156690); Cry32Mb1 (Accession #KC156704); Cry32Na1(Accession #KC156691); Cry32Oa1 (Accession #KC156703); Cry32Pa1(Accession #KC156705); Cry32Qa1 (Accession #KC156706); Cry32Ra1(Accession #KC156707); Cry32Sa1 (Accession #KC156709); Cry32Ta1(Accession #KC156710); Cry32Ua1 (Accession #KC156655); Cry33Aa1(Accession #AAL26871); Cry34Aa1 (Accession #AAG50341); Cry34Aa2(Accession #AAK64560); Cry34Aa3 (Accession #AAT29032); Cry34Aa4(Accession #AAT29030); Cry34Ab1 (Accession #AAG41671); Cry34Ac1(Accession #AAG50118); Cry34Ac2 (Accession #AAK64562); Cry34Ac3(Accession #AAT29029); Cry34Ba1 (Accession #AAK64565); Cry34Ba2(Accession #AAT29033); Cry34Ba3 (Accession #AAT29031); Cry35Aa1(Accession #AAG50342); Cry35Aa2 (Accession #AAK64561); Cry35Aa3(Accession #AAT29028); Cry35Aa4 (Accession #AAT29025); Cry35Ab1(Accession #AAG41672); Cry35Ab2 (Accession #AAK64563); Cry35Ab3(Accession #AY536891); Cry35Ac1 (Accession #AAG50117); Cry35Ba1(Accession #AAK64566); Cry35Ba2 (Accession #AAT29027); Cry35Ba3(Accession #AAT29026); Cry36Aa1 (Accession #AAK64558); Cry37Aa1(Accession #AAF76376); Cry38Aa1 (Accession #AAK64559); Cry39Aa1(Accession #BAB72016); Cry40Aa1 (Accession #BAB72018); Cry40Ba1(Accession #BAC77648); Cry40Ca1 (Accession #EU381045); Cry40Da1(Accession #ACF15199); Cry41Aa1 (Accession #BAD35157); Cry41Ab1(Accession #BAD35163); Cry41Ba1 (Accession #HM461871); Cry41Ba2(Accession #ZP 04099652); Cry42Aa1 (Accession #BAD35166); Cry43Aa1(Accession #BAD15301); Cry43Aa2 (Accession #BAD95474); Cry43Ba1(Accession #BAD15303); Cry43Ca1 (Accession #KC156676); Cry43Cb1(Accession #KC156695); Cry43Cc1 (Accession #KC156696); Cry43-like(Accession #BAD15305); Cry44Aa (Accession #BAD08532); Cry45Aa (Accession#BAD22577); Cry46Aa (Accession #BAC79010); Cry46Aa2 (Accession#BAG68906); Cry46Ab (Accession #BAD35170); Cry47Aa (Accession#AAY24695); Cry48Aa (Accession #CAJ18351); Cry48Aa2 (Accession#CAJ86545); Cry48Aa3 (Accession #CAJ86546); Cry48Ab (Accession#CAJ86548); Cry48Ab2 (Accession #CAJ86549); Cry49Aa (Accession#CAH56541); Cry49Aa2 (Accession #CAJ86541); Cry49Aa3 (Accession#CAJ86543); Cry49Aa4 (Accession #CAJ86544); Cry49Ab1 (Accession#CAJ86542); Cry50Aa1 (Accession #BAE86999); Cry50Ba1 (Accession#GU446675); Cry50Ba2 (Accession #GU446676); Cry51Aa1 (Accession#AB114444); Cry51Aa2 (Accession #GU570697); Cry52Aa1 (Accession#EF613489); Cry52Ba1 (Accession #FJ361760); Cry53Aa1 (Accession#EF633476); Cry53Ab1 (Accession #FJ361759); Cry54Aa1 (Accession#ACA52194); Cry54Aa2 (Accession #GQ140349); Cry54Ba1 (Accession#GU446677); Cry55Aa1 (Accession #ABW88932); Cry54Ab1 (Accession#JQ916908); Cry55Aa2 (Accession #AAE33526); Cry56Aa1 (Accession#ACU57499); Cry56Aa2 (Accession #GQ483512); Cry56Aa3 (Accession#JX025567); Cry57Aa1 (Accession #ANC87261); Cry58Aa1 (Accession#ANC87260); Cry59Ba1 (Accession #JN790647); Cry59Aa1 (Accession#ACR43758); Cry60Aa1 (Accession #ACU24782); Cry60Aa2 (Accession#EAO57254); Cry60Aa3 (Accession #EEM99278); Cry60Ba1 (Accession#GU810818); Cry60Ba2 (Accession #EAO57253); Cry60Ba3 (Accession#EEM99279); Cry61Aa1 (Accession #HM035087); Cry61Aa2 (Accession#HM132125); Cry61Aa3 (Accession #EEM19308); Cry62Aa1 (Accession#HM054509); Cry63Aa1 (Accession #BA144028); Cry64Aa1 (Accession#BAJQ5397); Cry65Aa1 (Accession #HM461868); Cry65Aa2 (Accession #ZP04123838); Cry66Aa1 (Accession #HM485581); Cry66Aa2 (Accession #ZP04099945); Cry67Aa1 (Accession #HM485582); Cry67Aa2 (Accession #ZP04148882); Cry68Aa1 (Accession #HQ113114); Cry69Aa1 (Accession#HQ401006); Cry69Aa2 (Accession #JQ821388); Cry69Ab1 (Accession#JN209957); Cry70Aa1 (Accession #JN646781); Cry70Ba1 (Accession#AD051070); Cry70Bb1 (Accession #EEL67276); Cry71Aa1 (Accession#JX025568); Cry72Aa1 (Accession #JX025569).

Examples of δ-endotoxins also include but are not limited to Cry1Aproteins of U.S. Pat. Nos. 5,880,275 and 7,858,849; a DIG-3 or DIG-11toxin (N-terminal deletion of α-helix 1 and/or α-helix 2 variants of Cryproteins such as Cry1A) of U.S. Pat. Nos. 8,304,604 and 8.304,605, Cry1Bof U.S. patent application Ser. No. 10/525,318; Cry1C of U.S. Pat. No.6,033,874; Cry1F of U.S. Pat. Nos. 5,188,960, 6,218,188; Cry1A/Fchimeras of U.S. Pat. Nos. 7,070,982; 6,962,705 and 6,713,063); a Cry2protein such as Cry2Ab protein of U.S. Pat. No. 7,064,249); a Cry3Aprotein including but not limited to an engineered hybrid insecticidalprotein (eHIP) created by fusing unique combinations of variable regionsand conserved blocks of at least two different Cry proteins (US PatentApplication Publication Number 2010/0017914); a Cry4 protein; a Cry5protein; a Cry6 protein; Cry8 proteins of U.S. Pat. Nos. 7,329,736,7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,378,499 and 7,462,760; aCry9 protein such as such as members of the Cry9A, Cry9B, Cry9C, Cry9D,Cry9E, and Cry9F families; a Cry15 protein of Naimov, et al., (2008)Applied and Environmental Microbiology 74:7145-7151; a Cry22, a Cry34Ab1protein of U.S. Pat. Nos. 6,127,180, 6,624,145 and 6,340,593; a CryET33and CryET34 protein of U.S. Pat. Nos. 6,248,535, 6,326,351, 6,399,330,6,949,626, 7,385,107 and 7,504,229; a CryET33 and CryET34 homologs of USPatent Publication Number 2006/0191034, 2012/0278954, and PCTPublication Number WO 2012/139004; a Cry35Ab1 protein of U.S. Pat. Nos.6,083,499, 6,548,291 and 6,340,593; a Cry46 protein, a Cry 51 protein, aCry binary toxin; a TIC901 or related toxin; TIC807 of US 2008/0295207;ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128 of PCT US2006/033867; TIC807 of US2040194351, TIC853 toxins of U.S. Pat. No.8,513,494, AXMI-027, AXMI-036, and AXMI-038 of U.S. Pat. No. 8,236,757;AXMI-031, AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. No. 7,923,602;AXMI-018, AXMI-020, and AXMI-021 of WO 2006/083891; AXMI-010 of WO2005/038032; AXMI-003 of WO 2005/021585; AXMI-008 of US 2004/0250311;AXMI-006 of US 2004/0216186; AXMI-007 of US 2004/0210965; AXMI-009 of US2004/0210964; AXMI-014 of US 2004/0197917; AXMI-004 of US 2004/0197916;AXMI-028 and AXMI-029 of WO 2006/119457; AXMI-007, AXMI-008,AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO 2004/074462;AXMI-150 of U.S. Pat. No. 8,084,416; AXMI-205 of US20110023184;AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-019, AXMI-044, AXMI-037,AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023, AXMI-041, AXMI-063,and AXMI-064 of US 2011/0263488; AXMI-R1 and related proteins of US2010/0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z and AXMI225z of WO2011/103248; AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228,AXMI229, AXMI230, and AXMI231 of WO11/103247; AXMI-115, AXMI-113,AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. Nos. 8,334,431; AXMI-001,AXMI-002, AXMI-030, AXMI-035, and AXMI-045 of US 2010/0298211; AXMI-066and AXMI-076 of US20090144852; AXMI128, AXMI130, AXMI131, AXMI133,AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148, AXMI149,AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158, AXMI162,AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, AXMI171, AXMI172,AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179, AXMI180,AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189 of U.S.Pat. No. 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082, AXMI091,AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102,AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111, AXMI112,AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122,AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164,AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of US 2010/0005543;AXMI221 of US20140196175; AXMI345 of US 20140373195; and Cry proteinssuch as Cry1A and Cry3A having modified proteolytic sites of U.S. Pat.No. 8,319,019; and a Cry1Ac, Cry2Aa and Cry1Ca toxin protein fromBacillus thuringiensis strain VBTS 2528 of US Patent ApplicationPublication Number 2011/0064710. Other Cry proteins are well known toone skilled in the art (see, Crickmore, et al., “Bacillus thuringiensistoxin nomenclature” (2011), atlifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/intro.html which can beaccessed on the world-wide web using the “www” prefix). The insecticidalactivity of Cry proteins is well known to one skilled in the art (forreview, see, van Frannkenhuyzen, (2009) J. Invert. Path. 101:1-16). Theuse of Cry proteins as transgenic plant traits is well known to oneskilled in the art and Cry-transgenic plants including but not limitedto Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2,Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A,mCry3A, Cry9c and CBI-Bt have received regulatory approval (see,Sanahuja, (2011) Plant Biotech Journal 9:283-300 and the CERA (2010) GMCrop Database Center for Environmental Risk Assessment (CERA), ILSIResearch Foundation, Washington, D.C. atcera-gmc.org/index.php?action=gm_crop_database which can be accessed onthe world-wide web using the “www” prefix). More than one pesticidalproteins well known to one skilled in the art can also be expressed inplants such as Vip3Ab & Cry1Fa (US2012/0317682), Cry1BE & Cry1F(US2012/0311746), Cry1CA & Cry1AB (US2012/0311745), Cry1F & CryCa(US2012/0317681), Cry1DA & Cry1BE (US2012/0331590), Cry1DA & Cry1Fa(US2012/0331589), Cry1AB & Cry1BE (US2012/0324606), and Cry1Fa & Cry2Aa,Cry1I or Cry1E (US2012/0324605). Pesticidal proteins also includeinsecticidal lipases including lipid acyl hydrolases of U.S. Pat. No.7,491,869, and cholesterol oxidases such as from Streptomyces (Purcellet al. (1993) Biochem Biophys Res Commun 15:1406-1413). Pesticidalproteins also include VIP (vegetative insecticidal proteins) toxins ofU.S. Pat. Nos. 5,877,012, 6,107,279, 6,137,033, 7,244,820, 7,615,686,and 8,237,020, and the like. Other VIP proteins are well known to oneskilled in the art (see,lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html which can beaccessed on the world-wide web using the “www” prefix). Pesticidalproteins also include toxin complex (TC) proteins, obtainable fromorganisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, U.S.Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have “stand alone”insecticidal activity and other TC proteins enhance the activity of thestand-alone toxins produced by the same given organism. The toxicity ofa “stand-alone” TC protein (from Photorhabdus, Xenorhabdus orPaenibacillus, for example) can be enhanced by one or more TC protein“potentiators” derived from a source organism of a different genus.There are three main types of TC proteins. As referred to herein, ClassA proteins (“Protein A”) are stand-alone toxins. Class B proteins(“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity ofClass A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 andXptA2. Examples of Class B proteins are TcaC, TcdB, XptB1Xb and XptC1Wi.Examples of Class C proteins are TccC, XptC1Xb and XptB1Wi. Pesticidalproteins also include spider, snake and scorpion venom proteins.Examples of spider venom peptides include but are not limited tolycotoxin-1 peptides and mutants thereof (U.S. Pat. No. 8,334,366).

(C) A polynucleotide encoding an insect-specific hormone or pheromonesuch as an ecdysteroid and juvenile hormone, a variant thereof, amimetic based thereon or an antagonist or agonist thereof. See, forexample, the disclosure by Hammock, et al., (1990) Nature 344:458, ofbaculovirus expression of cloned juvenile hormone esterase, aninactivator of juvenile hormone.

(D) A polynucleotide encoding an insect-specific peptide which, uponexpression, disrupts the physiology of the affected pest. For example,see the disclosures of, Regan, (1994) J. Biol. Chem. 269:9 (expressioncloning yields DNA coding for insect diuretic hormone receptor); Pratt,et al., (1989) Biochem. Biophys. Res. Comm. 163:1243 (an allostatin isidentified in Diploptera puntata); Chattopadhyay, et al., (2004)Critical Reviews in Microbiology 30(1):33-54; Zjawiony, (2004) J NatProd 67(2):300-310; Carlini and Grossi-de-Sa, (2002) Toxicon40(11):1515-1539; Ussuf, et al., (2001) Curr Sci. 80(7):847-853 andVasconcelos and Oliveira, (2004) Toxicon 44(4):385-403. See also, U.S.Pat. No. 5,266,317 to Tomalski, et al., who disclose genes encodinginsect-specific toxins.

(E) A polynucleotide encoding an enzyme responsible for ahyperaccumulation of a monoterpene, a sesquiterpene, a steroid,hydroxamic acid, a phenylpropanoid derivative or another non-proteinmolecule with insecticidal activity, including but not limited to7-epizingiberene synthase (US Patent Publication 20140157456).

(F) A polynucleotide encoding an enzyme involved in the modification,including the post-translational modification, of a biologically activemolecule; for example, a glycolytic enzyme, a proteolytic enzyme, alipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, ahydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, anelastase, a chitinase and a glucanase, whether natural or synthetic.See, PCT Application WO 1993/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase gene. DNA molecules whichcontain chitinase-encoding sequences can be obtained, for example, fromthe ATCC® under Accession Numbers 39637 and 67152. See also, Kramer, etal., (1993) Insect Biochem. Molec. Biol. 23:691, who teach thenucleotide sequence of a cDNA encoding tobacco hookworm chitinase andKawalleck, et al., (1993) Plant Molec. Biol. 21:673, who provide thenucleotide sequence of the parsley ubi4-2 polyubiquitin gene, and U.S.Pat. Nos. 6,563,020; 7,145,060 and 7,087,810.

(G) A polynucleotide encoding a molecule that stimulates signaltransduction. For example, see the disclosure by Botella, et al., (1994)Plant Molec. Biol. 24:757, of nucleotide sequences for mung beancalmodulin cDNA clones, and Griess, et al., (1994) Plant Physiol.104:1467, who provide the nucleotide sequence of a maize calmodulin cDNAclone.

(H) A polynucleotide encoding a hydrophobic moment peptide. See, PCTApplication WO 1995/16776 and U.S. Pat. No. 5,580,852 disclosure ofpeptide derivatives of Tachyplesin which inhibit fungal plant pathogens)and PCT Application WO 1995/18855 and U.S. Pat. No. 5,607,914 (teachessynthetic antimicrobial peptides that confer disease resistance).

(I) A polynucleotide encoding a membrane permease, a channel former or achannel blocker. For example, see the disclosure by Jaynes, et al.,(1993) Plant Sci. 89:43, of heterologous expression of a cecropin-betalytic peptide analog to render transgenic tobacco plants resistant toPseudomonas solanacearum.

(J) A gene encoding a viral-invasive protein or a complex toxin derivedtherefrom. For example, the accumulation of viral coat proteins intransformed plant cells imparts resistance to viral infection and/ordisease development effected by the virus from which the coat proteingene is derived, as well as by related viruses. See, Beachy, et al.,(1990) Ann. Rev. Phytopathol. 28:451. Coat protein-mediated resistancehas been conferred upon transformed plants against alfalfa mosaic virus,cucumber mosaic virus, tobacco streak virus, potato virus X, potatovirus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaicvirus. Id.

(K) A gene encoding an insect-specific antibody or an immunotoxinderived therefrom. Thus, an antibody targeted to a critical metabolicfunction in the insect gut would inactivate an affected enzyme, killingthe insect. Cf. Taylor, et al., Abstract #497, SEVENTH INT'L SYMPOSIUMON MOLECULAR PLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994)(enzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments).

(L) A gene encoding a virus-specific antibody. See, for example,Tavladoraki, et al., (1993) Nature 366:469, who show that transgenicplants expressing recombinant antibody genes are protected from virusattack.

(M) A polynucleotide encoding a developmental-arrestive protein producedin nature by a pathogen or a parasite. Thus, fungal endoalpha-1,4-D-polygalacturonases facilitate fungal colonization and plantnutrient release by solubilizing plant cell wallhomo-alpha-1,4-D-galacturonase. See, Lamb, et al., (1992) Bio/Technology10:1436. The cloning and characterization of a gene which encodes a beanendopolygalacturonase-inhibiting protein is described by Toubart, etal., (1992) Plant J. 2:367.

(N) A polynucleotide encoding a developmental-arrestive protein producedin nature by a plant. For example, Logemann, et al., (1992)Bio/Technology 10:305, have shown that transgenic plants expressing thebarley ribosome-inactivating gene have an increased resistance to fungaldisease.

(O) Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis related genes. Briggs, (1995) Current Biology5(2), Pieterse and Van Loon, (2004) Curr. Opin. Plant Bio. 7(4):456-64and Somssich, (2003) Cell 113(7):815-6.

(P) Antifungal genes (Cornelissen and Melchers, (1993) Pl. Physiol.101:709-712 and Parijs, et al., (1991) Planta 183:258-264 and Bushnell,et al., (1998) Can. J. of Plant Path. 20(2):137-149. Also see, U.S.patent application Ser. Nos. 09/950,933; 11/619,645; 11/657,710;11/748,994; 11/774,121 and U.S. Pat. Nos. 6,891,085 and 7,306,946. LysMReceptor-like kinases for the perception of chitin fragments as a firststep in plant defense response against fungal pathogens (US2012/0110696).

(Q) Detoxification genes, such as for fumonisin, beauvericin,moniliformin and zearalenone and their structurally related derivatives.For example, see, U.S. Pat. Nos. 5,716,820; 5,792,931; 5,798,255;5,846,812; 6,083,736; 6,538,177; 6,388,171 and 6,812,380.

(R) A polynucleotide encoding a Cystatin and cysteine proteinaseinhibitors. See, U.S. Pat. No. 7,205,453.

(S) Defensin genes. See, WO 2003/000863 and U.S. Pat. Nos. 6,911,577;6,855,865; 6,777,592 and 7,238,781.

(T) Genes conferring resistance to nematodes. See, e.g., PCT ApplicationWO 1996/30517; PCT Application WO 1993/19181, WO 2003/033651 and Urwin,et al., (1998) Planta 204:472-479, Williamson, (1999) Curr Opin PlantBio. 2(4):327-31; U.S. Pat. Nos. 6,284,948 and 7,301,069 and miR164genes (WO 2012/058266).

(U) Genes that confer resistance to Phytophthora Root Rot, such as theRps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7 and other Rps genes.See, for example, Shoemaker, et al., Phytophthora Root Rot ResistanceGene Mapping in Soybean, Plant Genome IV Conference, San Diego, Calif.(1995).

(V) Genes that confer resistance to Brown Stem Rot, such as described inU.S. Pat. No. 5,689,035 and incorporated by reference for this purpose.

(W) Genes that confer resistance to Colletotrichum, such as described inUS Patent Application Publication US 2009/0035765 and incorporated byreference for this purpose. This includes the Rcg locus that may beutilized as a single locus conversion.

2. Transgenes that Confer Resistance to a Herbicide, for Example:

(A) A polynucleotide encoding resistance to a herbicide that inhibitsthe growing point or meristem, such as an imidazolinone or asulfonylurea. Exemplary genes in this category code for mutant ALS andAHAS enzyme as described, for example, by Lee, et al., (1988) EMBO J.7:1241 and Miki, et al., (1990) Theor. Appl. Genet. 80:449,respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870;5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937 and5,378,824; U.S. patent application Ser. No. 11/683,737 and InternationalPublication WO 1996/33270.

(B) A polynucleotide encoding a protein for resistance to Glyphosate(resistance imparted by mutant 5-enolpyruvl-3-phosphikimate synthase(EPSP) and aroA genes, respectively) and other phosphono compounds suchas glufosinate (phosphinothricin acetyl transferase (PAT) andStreptomyces hygroscopicus phosphinothricin acetyl transferase (bar)genes), and pyridinoxy or phenoxy proprionic acids and cyclohexones(ACCase inhibitor-encoding genes). See, for example, U.S. Pat. No.4,940,835 to Shah, et al., which discloses the nucleotide sequence of aform of EPSPS which can confer glyphosate resistance. U.S. Pat. No.5,627,061 to Barry, et al., also describes genes encoding EPSPS enzymes.See also, U.S. Pat. Nos. 6,566,587; 6,338,961; 6,248,876 B1; 6,040,497;5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642;5,094,945, 4,940,835; 5,866,775; 6,225,114 B1; 6,130,366; 5,310,667;4,535,060; 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 E and5,491,288 and International Publications EP 1173580; WO 2001/66704; EP1173581 and EP 1173582, which are incorporated herein by reference forthis purpose. Glyphosate resistance is also imparted to plants thatexpress a gene encoding a glyphosate oxido-reductase enzyme as describedmore fully in U.S. Pat. Nos. 5,776,760 and 5,463,175, which areincorporated herein by reference for this purpose. In additionglyphosate resistance can be imparted to plants by the over expressionof genes encoding glyphosate N-acetyltransferase. See, for example, U.S.Pat. Nos. 7,462,481; 7,405,074 and US Patent Application PublicationNumber US 2008/0234130. A DNA molecule encoding a mutant aroA gene canbe obtained under ATCC® Accession Number 39256, and the nucleotidesequence of the mutant gene is disclosed in U.S. Pat. No. 4,769,061 toComai. EP Application Number 0 333 033 to Kumada, et al., and U.S. Pat.No. 4,975,374 to Goodman, et al., disclose nucleotide sequences ofglutamine synthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in EP ApplicationNumbers 0 242 246 and 0 242 236 to Leemans, et al.; De Greef, et al.,(1989) Bio/Technology 7:61, describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. See also, U.S. Pat. Nos. 5,969,213; 5,489,520;5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024;6,177,616 B1 and 5,879,903, which are incorporated herein by referencefor this purpose. Exemplary genes conferring resistance to phenoxyproprionic acids and cyclohexones, such as sethoxydim and haloxyfop, arethe Acc1-S1, Acc1-S2 and Acc1-S3 genes described by Marshall, et al.,(1992) Theor. Appl. Genet. 83:435.

(C) A polynucleotide encoding a protein for resistance to herbicide thatinhibits photosynthesis, such as a triazine (psbA and gs+genes) and abenzonitrile (nitrilase gene). Przibilla, et al., (1991) Plant Cell3:169, describe the transformation of Chlamydomonas with plasmidsencoding mutant psbA genes. Nucleotide sequences for nitrilase genes aredisclosed in U.S. Pat. No. 4,810,648 to Stalker and DNA moleculescontaining these genes are available under ATCC® Accession Numbers53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes, et al., (1992) Biochem.J. 285:173.

(D) A polynucleotide encoding a protein for resistance to Acetohydroxyacid synthase, which has been found to make plants that express thisenzyme resistant to multiple types of herbicides, has been introducedinto a variety of plants (see, e.g., Hattori, et al., (1995) Mol GenGenet. 246:419). Other genes that confer resistance to herbicidesinclude: a gene encoding a chimeric protein of rat cytochrome P4507A1and yeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., (1994)Plant Physiol 106:17), genes for glutathione reductase and superoxidedismutase (Aono, et al., (1995) Plant Cell Physiol 36:1687) and genesfor various phosphotransferases (Datta, et al., (1992) Plant Mol Biol20:619).

(E) A polynucleotide encoding resistance to a herbicide targetingProtoporphyrinogen oxidase (protox) which is necessary for theproduction of chlorophyll. The protox enzyme serves as the target for avariety of herbicidal compounds. These herbicides also inhibit growth ofall the different species of plants present, causing their totaldestruction. The development of plants containing altered protoxactivity which are resistant to these herbicides are described in U.S.Pat. Nos. 6,288,306 B1; 6,282,837 B1 and 5,767,373 and InternationalPublication WO 2001/12825.

(F) The aad-1 gene (originally from Sphingobium herbicidovorans) encodesthe aryloxyalkanoate dioxygenase (AAD-1) protein. The trait conferstolerance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate(commonly referred to as “fop” herbicides such as quizalofop)herbicides. The aad-1 gene, itself, for herbicide tolerance in plantswas first disclosed in WO 2005/107437 (see also, US 2009/0093366). Theaad-12 gene, derived from Delftia acidovorans, which encodes thearyloxyalkanoate dioxygenase (AAD-12) protein that confers tolerance to2,4-dichlorophenoxyacetic acid and pyridyloxyacetate herbicides bydeactivating several herbicides with an aryloxyalkanoate moiety,including phenoxy auxin (e.g., 2,4-D, MCPA), as well as pyridyloxyauxins (e.g., fluroxypyr, triclopyr).

(G) A polynucleotide encoding a herbicide resistant dicambamonooxygenase disclosed in US Patent Application Publication2003/0135879 for imparting dicamba tolerance;

(H) A polynucleotide molecule encoding bromoxynil nitrilase (Bxn)disclosed in U.S. Pat. No. 4,810,648 for imparting bromoxynil tolerance;

(I) A polynucleotide molecule encoding phytoene (crtl) described inMisawa, et al., (1993) Plant J. 4:833-840 and in Misawa, et al., (1994)Plant J. 6:481-489 for norflurazon tolerance.

3. Transgenes that Confer or Contribute to an Altered GrainCharacteristic

Such as:

(A) Altered fatty acids, for example, by

(1) Down-regulation of stearoyl-ACP to increase stearic acid content ofthe plant. See, Knultzon, et al., (1992) Proc. Natl. Acad. Sci. USA89:2624 and WO 1999/64579 (Genes to Alter Lipid Profiles in Corn).

(2) Elevating oleic acid via FAD-2 gene modification and/or decreasinglinolenic acid via FAD-3 gene modification (see, U.S. Pat. Nos.6,063,947; 6,323,392; 6,372,965 and WO 1993/11245).

(3) Altering conjugated linolenic or linoleic acid content, such as inWO 2001/12800.

(4) Altering LEC1, AGP, Dek1, Superal1, mi1 ps, various Ipa genes suchas Ipa1, Ipa3, hpt or hggt. For example, see, WO 2002/42424, WO1998/22604, WO 2003/011015, WO 2002/057439, WO 2003/011015, U.S. Pat.Nos. 6,423,886, 6,197,561, 6,825,397 and US Patent ApplicationPublication Numbers US 2003/0079247, US 2003/0204870 and Rivera-Madrid,et al., (1995) Proc. Natl. Acad. Sci. 92:5620-5624.

(5) Genes encoding delta-8 desaturase for making long-chainpolyunsaturated fatty acids (U.S. Pat. Nos. 8,058,571 and 8,338,152),delta-9 desaturase for lowering saturated fats (U.S. Pat. No.8,063,269), Primula Δ6-desaturase for improving omega-3 fatty acidprofiles.

(6) Isolated nucleic acids and proteins associated with lipid and sugarmetabolism regulation, in particular, lipid metabolism protein (LMP)used in methods of producing transgenic plants and modulating levels ofseed storage compounds including lipids, fatty acids, starches or seedstorage proteins and use in methods of modulating the seed size, seednumber, seed weights, root length and leaf size of plants (EP 2404499).

(7) Altering expression of a High-Level Expression of Sugar-Inducible 2(HSI2) protein in the plant to increase or decrease expression of HSI2in the plant. Increasing expression of HSI2 increases oil content whiledecreasing expression of HSI2 decreases abscisic acid sensitivity and/orincreases drought resistance (US Patent Application Publication Number2012/0066794).

(8) Expression of cytochrome b5 (Cb5) alone or with FAD2 to modulate oilcontent in plant seed, particularly to increase the levels of omega-3fatty acids and improve the ratio of omega-6 to omega-3 fatty acids (USPatent Application Publication Number 2011/0191904).

(9) Nucleic acid molecules encoding wrinkled1-like polypeptides formodulating sugar metabolism (U.S. Pat. No. 8,217,223).

(B) Altered phosphorus content, for example, by the

(1) Introduction of a phytase-encoding gene would enhance breakdown ofphytate, adding more free phosphate to the transformed plant. Forexample, see, Van Hartingsveldt, et al., (1993) Gene 127:87, for adisclosure of the nucleotide sequence of an Aspergillus niger phytasegene.

(2) Modulating a gene that reduces phytate content. In maize, this, forexample, could be accomplished, by cloning and then re-introducing DNAassociated with one or more of the alleles, such as the LPA alleles,identified in maize mutants characterized by low levels of phytic acid,such as in WO 2005/113778 and/or by altering inositol kinase activity asin WO 2002/059324, US Patent Application Publication Number2003/0009011, WO 2003/027243, US Patent Application Publication Number2003/0079247, WO 1999/05298, U.S. Pat. Nos. 6,197,561, 6,291,224,6,391,348, WO 2002/059324, US Patent Application Publication Number2003/0079247, WO 1998/45448, WO 1999/55882, WO 2001/04147.

(C) Altered carbohydrates affected, for example, by altering a gene foran enzyme that affects the branching pattern of starch or, a genealtering thioredoxin such as NTR and/or TRX (see, U.S. Pat. No.6,531,648. which is incorporated by reference for this purpose) and/or agamma zein knock out or mutant such as cs27 or TUSC27 or en27 (see, U.S.Pat. No. 6,858,778 and US Patent Application Publication Number2005/0160488, US Patent Application Publication Number 2005/0204418,which are incorporated by reference for this purpose). See, Shiroza, etal., (1988) J. Bacteriol. 170:810 (nucleotide sequence of Streptococcusmutant fructosyltransferase gene), Steinmetz, et al., (1985) Mol. Gen.Genet. 200:220 (nucleotide sequence of Bacillus subtilis levansucrasegene), Pen, et al., (1992) Bio/Technology 10:292 (production oftransgenic plants that express Bacillus licheniformis alpha-amylase),Elliot, et al., (1993) Plant Molec. Biol. 21:515 (nucleotide sequencesof tomato invertase genes), Sogaard, et al., (1993) J. Biol. Chem.268:22480 (site-directed mutagenesis of barley alpha-amylase gene) andFisher, et al., (1993) Plant Physiol. 102:1045 (maize endosperm starchbranching enzyme II), WO 1999/10498 (improved digestibility and/orstarch extraction through modification of UDP-D-xylose 4-epimerase,Fragile 1 and 2, Ref 1, HCHL, C4H), U.S. Pat. No. 6,232,529 (method ofproducing high oil seed by modification of starch levels (AGP)). Thefatty acid modification genes mentioned herein may also be used toaffect starch content and/or composition through the interrelationshipof the starch and oil pathways.

(D) Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. For example, see, U.S. Pat. No. 6,787,683,US Patent Application Publication Number 2004/0034886 and WO 2000/68393involving the manipulation of antioxidant levels and WO 2003/082899through alteration of a homogentisate geranyl geranyl transferase(hggt).

(E) Altered essential seed amino acids. For example, see, U.S. Pat. No.6,127,600 (method of increasing accumulation of essential amino acids inseeds), U.S. Pat. No. 6,080,913 (binary methods of increasingaccumulation of essential amino acids in seeds), U.S. Pat. No. 5,990,389(high lysine), WO 1999/40209 (alteration of amino acid compositions inseeds), WO 1999/29882 (methods for altering amino acid content ofproteins), U.S. Pat. No. 5,850,016 (alteration of amino acidcompositions in seeds), WO 1998/20133 (proteins with enhanced levels ofessential amino acids), U.S. Pat. No. 5,885,802 (high methionine), U.S.Pat. No. 5,885,801 (high threonine), U.S. Pat. No. 6,664,445 (plantamino acid biosynthetic enzymes), U.S. Pat. No. 6,459,019 (increasedlysine and threonine), U.S. Pat. No. 6,441,274 (plant tryptophansynthase beta subunit), U.S. Pat. No. 6,346,403 (methionine metabolicenzymes), U.S. Pat. No. 5,939,599 (high sulfur), U.S. Pat. No. 5,912,414(increased methionine), WO 1998/56935 (plant amino acid biosyntheticenzymes), WO 1998/45458 (engineered seed protein having higherpercentage of essential amino acids), WO 1998/42831 (increased lysine),U.S. Pat. No. 5,633,436 (increasing sulfur amino acid content), U.S.Pat. No. 5,559,223 (synthetic storage proteins with defined structurecontaining programmable levels of essential amino acids for improvementof the nutritional value of plants), WO 1996/01905 (increasedthreonine), WO 1995/15392 (increased lysine), US Patent ApplicationPublication Number 2003/0163838, US Patent Application PublicationNumber 2003/0150014, US Patent Application Publication Number2004/0068767, U.S. Pat. No. 6,803,498, WO 2001/79516.

4. Genes that Control Male-Sterility:

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar, et al., and chromosomal translocationsas described by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen, et al., U.S. Pat. No. 5,432,068,describe a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility; silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

(A) Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT (WO 2001/29237).

(B) Introduction of various stamen-specific promoters (WO 1992/13956, WO1992/13957).

(C) Introduction of the barnase and the barstar gene (Paul, et al.,(1992) Plant Mol. Biol. 19:611-622).

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. Nos. 5,859,341; 6,297,426; 5,478,369;5,824,524; 5,850,014 and 6,265,640, all of which are hereby incorporatedby reference.

5. Genes that Create a Site for Site Specific DNA Integration.

This includes the introduction of FRT sites that may be used in theFLP/FRT system and/or Lox sites that may be used in the Cre/Loxp system.For example, see, Lyznik, et al., (2003) Plant Cell Rep 21:925-932 andWO 1999/25821, which are hereby incorporated by reference. Other systemsthat may be used include the Gin recombinase of phage Mu (Maeser, etal., (1991) Vicki Chandler, The Maize Handbook ch. 118 (Springer-Verlag1994), the Pin recombinase of E. coli (Enomoto, et al., 1983) and theR/RS system of the pSRi plasmid (Araki, et al., 1992).

6. Genes that Affect Abiotic Stress Resistance

Including but not limited to flowering, ear and seed development,enhancement of nitrogen utilization efficiency, altered nitrogenresponsiveness, drought resistance or tolerance, cold resistance ortolerance and salt resistance or tolerance and increased yield understress.

(A) For example, see: WO 2000/73475 where water use efficiency isaltered through alteration of malate; U.S. Pat. Nos. 5,892,009,5,965,705, 5,929,305, 5,891,859, 6,417,428, 6,664,446, 6,706,866,6,717,034, 6,801,104, WO 2000/060089, WO 2001/026459, WO 2001/035725, WO2001/034726, WO 2001/035727, WO 2001/036444, WO 2001/036597, WO2001/036598, WO 2002/015675, WO 2002/017430, WO 2002/077185, WO2002/079403, WO 2003/013227, WO 2003/013228, WO 2003/014327, WO2004/031349, WO 2004/076638, WO 199809521.

(B) WO 199938977 describing genes, including CBF genes and transcriptionfactors effective in mitigating the negative effects of freezing, highsalinity and drought on plants, as well as conferring other positiveeffects on plant phenotype.

(C) US Patent Application Publication Number 2004/0148654 and WO2001/36596 where abscisic acid is altered in plants resulting inimproved plant phenotype such as increased yield and/or increasedtolerance to abiotic stress.

(D) WO 2000/006341, WO 2004/090143, U.S. Pat. Nos. 7,531,723 and6,992,237 where cytokinin expression is modified resulting in plantswith increased stress tolerance, such as drought tolerance, and/orincreased yield. Also see, WO 2002/02776, WO 2003/052063, JP2002/281975, U.S. Pat. No. 6,084,153, WO 2001/64898, U.S. Pat. No.6,177,275 and U.S. Pat. No. 6,107,547 (enhancement of nitrogenutilization and altered nitrogen responsiveness).

(E) For ethylene alteration, see, US Patent Application PublicationNumber 2004/0128719, US Patent Application Publication Number2003/0166197 and WO 2000/32761.

(F) For plant transcription factors or transcriptional regulators ofabiotic stress, see, e.g., US Patent Application Publication Number2004/0098764 or US Patent Application Publication Number 2004/0078852.

(G) Genes that increase expression of vacuolar pyrophosphatase such asAVP1 (U.S. Pat. No. 8,058,515) for increased yield; nucleic acidencoding a HSFA4 or a HSFA5 (Heat Shock Factor of the class A4 or A5)polypeptides, an oligopeptide transporter protein (OPT4-like)polypeptide; a plastochron2-like (PLA2-like) polypeptide or a Wuschelrelated homeobox 1-like (WOX1-like) polypeptide (U. Patent ApplicationPublication Number US 2011/0283420).

(H) Down regulation of polynucleotides encoding poly (ADP-ribose)polymerase (PARP) proteins to modulate programmed cell death (U.S. Pat.No. 8,058,510) for increased vigor.

(I) Polynucleotide encoding DTP21 polypeptides for conferring droughtresistance (US Patent Application Publication Number US 2011/0277181).

(J) Nucleotide sequences encoding ACC Synthase 3 (ACS3) proteins formodulating development, modulating response to stress, and modulatingstress tolerance (US Patent Application Publication Number US2010/0287669).

(K) Polynucleotides that encode proteins that confer a drought tolerancephenotype (DTP) for conferring drought resistance (WO 2012/058528).

(L) Tocopherol cyclase (TC) genes for conferring drought and salttolerance (US Patent Application Publication Number 2012/0272352).

(M) CAAX amino terminal family proteins for stress tolerance (U.S. Pat.No. 8,338,661).

(N) Mutations in the SAL1 encoding gene have increased stress tolerance,including increased drought resistant (US Patent Application PublicationNumber 2010/0257633).

(O) Expression of a nucleic acid sequence encoding a polypeptideselected from the group consisting of: GRF polypeptide, RAA1-likepolypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptideincreasing yield-related traits (US Patent Application PublicationNumber 2011/0061133).

(P) Modulating expression in a plant of a nucleic acid encoding a ClassIII Trehalose Phosphate Phosphatase (TPP) polypeptide for enhancingyield-related traits in plants, particularly increasing seed yield (USPatent Application Publication Number 2010/0024067).

Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth and/or plantstructure, can be introduced or introgressed into plants, see e.g., WO1997/49811 (LHY), WO 1998/56918 (ESD4), WO 1997/10339 and U.S. Pat. No.6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO 1996/14414 (CON), WO1996/38560, WO 2001/21822 (VRN1), WO 2000/44918 (VRN2), WO 1999/49064(GI), WO 2000/46358 (FR1), WO 1997/29123, U.S. Pat. No. 6,794,560,6,307,126 (GAI), WO 1999/09174 (D8 and Rht) and WO 2004/076638 and WO2004/031349 (transcription factors).

7. Genes that Confer Increased Yield

(A) A transgenic crop plant transformed by a1-AminoCyclopropane-1-Carboxylate Deaminase-like Polypeptide (ACCDP)coding nucleic acid, wherein expression of the nucleic acid sequence inthe crop plant results in the plant's increased root growth, and/orincreased yield, and/or increased tolerance to environmental stress ascompared to a wild type variety of the plant (U.S. Pat. No. 8,097,769).

(B) Over-expression of maize zinc finger protein gene (Zm-ZFP1) using aseed preferred promoter has been shown to enhance plant growth, increasekernel number and total kernel weight per plant (US Patent ApplicationPublication Number 2012/0079623).

(C) Constitutive over-expression of maize lateral organ boundaries (LOB)domain protein (Zm-LOBDP1) has been shown to increase kernel number andtotal kernel weight per plant (US Patent Application Publication Number2012/0079622).

(D) Enhancing yield-related traits in plants by modulating expression ina plant of a nucleic acid encoding a VIM1 (Variant in Methylation1)-like polypeptide or a VTC2-like (GDP-L-galactose phosphorylase)polypeptide or a DUF1685 polypeptide or an ARF6-like (Auxin ResponsiveFactor) polypeptide (WO 2012/038893).

(E) Modulating expression in a plant of a nucleic acid encoding aSte20-like polypeptide or a homologue thereof gives plants havingincreased yield relative to control plants (EP 2431472).

(F) Genes encoding nucleoside diphosphatase kinase (NDK) polypeptidesand homologs thereof for modifying the plant's root architecture (USPatent Application Publication Number 2009/0064373).

8. Genes that Confer Plant Digestibility.

(A) Altering the level of xylan present in the cell wall of a plant bymodulating expression of xylan synthase (U.S. Pat. No. 8,173,866).

In some embodiment the stacked trait may be a trait or event that hasreceived regulatory approval including but not limited to the events inTable 4A -4F.

TABLE 4A Helianthus annuus Sunflower Event Company Description X81359BASF Inc. Tolerance to imidazolinone herbicides by selection of anaturally occurring mutant.

TABLE 4B Oryza sativa Rice Event Company Description CL121, CL141, CFX51BASF Inc. Tolerance to the imidazolinone herbicide, imazethapyr, inducedby chemical mutagenesis of the acetolactate synthase (ALS) enzyme usingethyl methanesulfonate (EMS). IMINTA-1, IMINTA-4 BASF Inc. Tolerance toimidazolinone herbicides induced by chemical mutagenesis of theacetolactate synthase (ALS) enzyme using sodium azide. LLRICE06,LLRICE62 Aventis CropScience Glufosinate ammonium herbicide tolerantrice produced by inserting a modified phosphinothricin acetyltransferase(PAT) encoding gene from the soil bacterium Streptomyces hygroscopicus).LLRICE601 Bayer CropScience Glufosinate ammonium herbicide tolerant rice(Aventis produced by inserting a modified phosphinothricinCropScience(AgrEvo)) acetyltransferase (PAT) encoding gene from the soilbacterium Streptomyces hygroscopicus). PWC16 BASF Inc. Tolerance to theimidazolinone herbicide, imazethapyr, induced by chemical mutagenesis ofthe acetolactate synthase (ALS) enzyme using ethyl methanesulfonate(EMS).

TABLE 4C Glycine max L. Soybean Event Company Description A2704-12,A2704-21, Bayer CropScience Glufosinate ammonium herbicide tolerantsoybean A5547-35 (Aventis CropScience produced by inserting a modifiedphosphinothricin (AgrEvo)) acetyltransferase (PAT) encoding gene fromthe soil bacterium Streptomyces viridochromogenes. A5547-127 BayerCropScience Glufosinate ammonium herbicide tolerant soybean (AventisCropScience produced by inserting a modified phosphinothricin (AgrEvo))acetyltransferase (PAT) encoding gene from the soil bacteriumStreptomyces viridochromogenes. BPS-CV127-9 BASF Inc. The introducedcsr1-2 gene from Arabidopsis thaliana encodes an acetohydroxyacidsynthase protein that confers tolerance to imidazolinone herbicides dueto a point mutation that results in a single amino acid substitution inwhich the serine residue at position 653 is replaced by asparagine(S653N). DP-305423 Pioneer Hi-Bred High oleic acid soybean produced byinserting International Inc. additional copies of a portion of theomega-6 desaturase encoding gene, gm-fad2-1 resulting in silencing ofthe endogenous omega-6 desaturase gene (FAD2-1). DP356043 PioneerHi-Bred Soybean event with two herbicide tolerance genes: InternationalInc. glyphosate N-acetlytransferase, which detoxifies glyphosate, and amodified acetolactate synthase (ALS) gene which is tolerant toALS-inhibiting herbicides. G94-1, G94-19, G168 DuPont Canada High oleicacid soybean produced by inserting a Agricultural Products second copyof the fatty acid desaturase (GmFad2-1) encoding gene from soybean,which resulted in “silencing” of the endogenous host gene. GTS 40-3-2Monsanto Company Glyphosate tolerant soybean variety produced byinserting a modified 5-enolpyruvylshikimate-3- phosphate synthase(EPSPS) encoding gene from the soil bacterium Agrobacterium tumefaciens.GU262 Bayer CropScience Glufosinate ammonium herbicide tolerant soybean(Aventis produced by inserting a modified phosphinothricinCropScience(AgrEvo)) acetyltransferase (PAT) encoding gene from the soilbacterium Streptomyces viridochromogenes. MON87701 Monsanto CompanyResistance to Lepidopteran pests of soybean including velvetbeancaterpillar (Anticarsia gemmatalis) and soybean looper (Pseudoplusiaincludens). MON87701 × Monsanto Company Glyphosate herbicide tolerancethrough expression MON89788 of the EPSPS encoding gene from A.tumefaciens strain CP4, and resistance to Lepidopteran pests of soybeanincluding velvetbean caterpillar (Anticarsia gemmatalis) and soybeanlooper (Pseudoplusia includens) via expression of the Cry1Ac encodinggene from B. thuringiensis. MON89788 Monsanto CompanyGlyphosate-tolerant soybean produced by inserting a modified5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding aroA(epsps) gene from Agrobacterium tumefaciens CP4. OT96-15 Agriculture &Agri-Food Low linolenic acid soybean produced through Canada traditionalcross-breeding to incorporate the novel trait from a naturally occurringfan1 gene mutant that was selected for low linolenic acid. W62, W98Bayer CropScience Glufosinate ammonium herbicide tolerant soybean(Aventis produced by inserting a modified phosphinothricinCropScience(AgrEvo)) acetyltransferase (PAT) encoding gene from the soilbacterium Streptomyces hygroscopicus.

TABLE 4D Triticum aestivum Wheat Event Company Description AP205CL BASFInc. Selection for a mutagenized version of the enzyme acetohydroxyacidsynthase (AHAS), also known as acetolactate synthase (ALS) oracetolactate pyruvate- lyase. AP602CL BASF Inc. Selection for amutagenized version of the enzyme acetohydroxyacid synthase (AHAS), alsoknown as acetolactate synthase (ALS) or acetolactate pyruvate- lyase.BW255-2, BW238-3 BASF Inc. Selection for a mutagenized version of theenzyme acetohydroxyacid synthase (AHAS), also known as acetolactatesynthase (ALS) or acetolactate pyruvate- lyase. BW7 BASF Inc. Toleranceto imidazolinone herbicides induced by chemical mutagenesis of theacetohydroxyacid synthase (AHAS) gene using sodium azide. MON71800Monsanto Company Glyphosate tolerant wheat variety produced by insertinga modified 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encodinggene from the soil bacterium Agrobacterium tumefaciens, strain CP4.SWP965001 Cyanamid Crop Selection for a mutagenized version of theenzyme Protection acetohydroxyacid synthase (AHAS), also known asacetolactate synthase (ALS) or acetolactate pyruvate- lyase. Teal 11ABASF Inc. Selection for a mutagenized version of the enzymeacetohydroxyacid synthase (AHAS), also known as acetolactate synthase(ALS) or acetolactate pyruvate- lyase.

TABLE 4E Medicago sativa Alfalfa Event Company Description J101,Monsanto Company Glyphosate herbicide tolerant alfalfa J163 and Forage(lucerne) produced by inserting a gene Genetics encoding the enzymeInternational 5-enolypyruvylshikimate-3-phosphate synthase (EPSPS) fromthe CP4 strain of Agrobacterium tumefaciens.

TABLE 4F Zea mays L. Maize Event Company Description 176 Syngenta Seeds,Inc. Insect-resistant maize produced by inserting the Cry1Ab gene fromBacillus thuringiensis subsp. kurstaki. The genetic modification affordsresistance to attack by the European corn borer (ECB). 3751IR PioneerHi-Bred Selection of somaclonal variants by culture of InternationalInc. embryos on imidazolinone containing media. 676, 678, 680 PioneerHi-Bred Male-sterile and glufosinate ammonium herbicide InternationalInc. tolerant maize produced by inserting genes encoding DNA adeninemethylase and phosphinothricin acetyltransferase (PAT) from Escherichiacoli and Streptomyces viridochromogenes, respectively. B16 (DLL25)Dekalb Genetics Glufosinate ammonium herbicide tolerant maizeCorporation produced by inserting the gene encoding phosphinothricinacetyltransferase (PAT) from Streptomyces hygroscopicus. BT11 (X4334CBR,Syngenta Seeds, Inc. Insect-resistant and herbicide tolerant maizeX4734CBR) produced by inserting the Cry1Ab gene from Bacillusthuringiensis subsp. kurstaki, and the phosphinothricinN-acetyltransferase (PAT) encoding gene from S. viridochromogenes. BT11× GA21 Syngenta Seeds, Inc. Stacked insect resistant and herbicidetolerant maize produced by conventional cross breeding of parental linesBT11 (OECD unique identifier: SYN- BTO11-1) and GA21 (OECD uniqueidentifier: MON-OOO21-9). BT11 × MIR162 × Syngenta Seeds, Inc.Resistance to Coleopteran pests, particularly corn MIR604 × GA21rootworm pests (Diabrotica spp.) and several Lepidopteran pests of corn,including European corn borer (ECB, Ostrinia nubilalis), corn earworm(CEW, Helicoverpa zea), fall army worm (FAW, Spodoptera frugiperda), andblack cutworm (BCW, Agrotis ipsilon); tolerance to glyphosate andglufosinate-ammonium containing herbicides. BT11 × MIR162 SyngentaSeeds, Inc. Stacked insect resistant and herbicide tolerant maizeproduced by conventional cross breeding of parental lines BT11 (OECDunique identifier: SYN- BTO11-1) and MIR162 (OECD unique identifier:SYN-IR162-4). Resistance to the European Corn Borer and tolerance to theherbicide glufosinate ammonium (Liberty) is derived from BT11, whichcontains the Cry1Ab gene from Bacillus thuringiensis subsp. kurstaki,and the phosphinothricin N-acetyltransferase (PAT) encoding gene from S.viridochromogenes. Resistance to other Lepidopteran pests, including H.zea, S. frugiperda, A. ipsilon, and S. albicosta, is derived fromMIR162, which contains the vip3Aa gene from Bacillus thuringiensisstrain AB88. BT11 × MIR162 × Syngenta Seeds, Inc. Bacillus thuringiensisCry1Ab delta-endotoxin MIR604 protein and the genetic material necessaryfor its production (via elements of vector pZO1502) in Event Bt11 corn(OECD Unique Identifier: SYN- BTO11-1) × Bacillus thuringiensis Vip3Aa20insecticidal protein and the genetic material necessary for itsproduction (via elements of vector pNOV1300) in Event MIR162 maize (OECDUnique Identifier: SYN-IR162-4) × modified Cry3A protein and the geneticmaterial necessary for its production (via elements of vector pZM26) inEvent MIR604 corn (OECD Unique Identifier: SYN- IR6O4-5). CBH-351Aventis CropScience Insect-resistant and glufosinate ammonium herbicidetolerant maize developed by inserting genes encoding Cry9C protein fromBacillus thuringiensis subsp tolworthi and phosphinothricinacetyltransferase (PAT) from Streptomyces hygroscopicus. DAS-06275-8 DOWAgroSciences LLC Lepidopteran insect resistant and glufosinate ammoniumherbicide-tolerant maize variety produced by inserting the Cry1F genefrom Bacillus thuringiensis var aizawai and the phosphinothricinacetyltransferase (PAT) from Streptomyces hygroscopicus. BT11 × MIR604Syngenta Seeds, Inc. Stacked insect resistant and herbicide tolerantmaize produced by conventional cross breeding of parental lines BT11(OECD unique identifier: SYN- BTO11-1) and MIR604 (OECD uniqueidentifier: SYN-IR6O5-5). Resistance to the European Corn Borer andtolerance to the herbicide glufosinate ammonium (Liberty) is derivedfrom BT11, which contains the Cry1Ab gene from Bacillus thuringiensissubsp. kurstaki, and the phosphinothricin N-acetyltransferase (PAT)encoding gene from S. viridochromogenes. Corn rootworm-resistance isderived from MIR604 which contains the mCry3A gene from Bacillusthuringiensis. BT11 × MIR604 × GA21 Syngenta Seeds, Inc. Stacked insectresistant and herbicide tolerant maize produced by conventional crossbreeding of parental lines BT11 (OECD unique identifier: SYN- BTO11-1),MIR604 (OECD unique identifier: SYN- IR6O5-5) and GA21 (OECD uniqueidentifier: MON-OOO21-9). Resistance to the European Corn Borer andtolerance to the herbicide glufosinate ammonium (Liberty) is derivedfrom BT11, which contains the Cry1Ab gene from Bacillus thuringiensissubsp. kurstaki, and the phosphinothricin N-acetyltransferase (PAT)encoding gene from S. viridochromogenes. Corn rootworm-resistance isderived from MIR604 which contains the mCry3A gene from Bacillusthuringiensis. Tolerance to glyphosate herbicide is derived from GA21which contains a a modified EPSPS gene from maize. DAS-59122-7 DOWAgroSciences LLC Corn rootworm-resistant maize produced by and PioneerHi-Bred inserting the Cry34Ab1 and Cry35Ab1 genes from InternationalInc. Bacillus thuringiensis strain PS149B1. The PAT encoding gene fromStreptomyces viridochromogenes was introduced as a selectable marker.DAS-59122-7 × TC1507 × DOW AgroSciences LLC Stacked insect resistant andherbicide tolerant NK603 and Pioneer Hi-Bred maize produced byconventional cross breeding of International Inc. parental linesDAS-59122-7 (OECD unique identifier: DAS-59122-7) and TC1507 (OECDunique identifier: DAS-O15O7-1) with NK603 (OECD unique identifier:MON-OO6O3-6). Corn rootworm-resistance is derived from DAS-59122-7 whichcontains the Cry34Ab1 and Cry35Ab1 genes from Bacillus thuringiensisstrain PS149B1. Lepidopteran resistance and tolerance to glufosinateammonium herbicide is derived from TC1507. Tolerance to glyphosateherbicide is derived from NK603. DBT418 Dekalb Genetics Insect-resistantand glufosinate ammonium Corporation herbicide tolerant maize developedby inserting genes encoding Cry1AC protein from Bacillus thuringiensissubsp kurstaki and phosphinothricin acetyltransferase (PAT) fromStreptomyces hygroscopicus MIR604 × GA21 Syngenta Seeds, Inc. Stackedinsect resistant and herbicide tolerant maize produced by conventionalcross breeding of parental lines MIR604 (OECD unique identifier:SYN-IR6O5-5) and GA21 (OECD unique identifier: MON-OOO21-9). Cornrootworm-resistance is derived from MIR604 which contains the mCry3Agene from Bacillus thuringiensis. Tolerance to glyphosate herbicide isderived from GA21. MON80100 Monsanto Company Insect-resistant maizeproduced by inserting the Cry1Ab gene from Bacillus thuringiensis subsp.kurstaki. The genetic modification affords resistance to attack by theEuropean corn borer (ECB). MON802 Monsanto Company Insect-resistant andglyphosate herbicide tolerant maize produced by inserting the genesencoding the Cry1Ab protein from Bacillus thuringiensis and the5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from A. tumefaciensstrain CP4. MON809 Pioneer Hi-Bred Resistance to European corn borer(Ostrinia International Inc. nubilalis) by introduction of a syntheticCry1Ab gene. Glyphosate resistance via introduction of the bacterialversion of a plant enzyme, 5-enolpyruvyl shikimate-3-phosphate synthase(EPSPS). MON810 Monsanto Company Insect-resistant maize produced byinserting a truncated form of the Cry1Ab gene from Bacillusthuringiensis subsp. kurstaki HD-1. The genetic modification affordsresistance to attack by the European corn borer (ECB). MON810 × LY038Monsanto Company Stacked insect resistant and enhanced lysine contentmaize derived from conventional cross- breeding of the parental linesMON810 (OECD identifier: MON-OO81O-6) and LY038 (OECD identifier:REN-OOO38-3). MON810 × MON88017 Monsanto Company Stacked insectresistant and glyphosate tolerant maize derived from conventionalcross-breeding of the parental lines MON810 (OECD identifier:MON-OO81O-6) and MON88017 (OECD identifier: MON-88O17-3). European cornborer (ECB) resistance is derived from a truncated form of the Cry1Abgene from Bacillus thuringiensis subsp. kurstaki HD-1 present in MON810.Corn rootworm resistance is derived from the Cry3Bb1 gene from Bacillusthuringiensis subspecies kumamotoensis strain EG4691 present inMON88017. Glyphosate tolerance is derived from a5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene fromAgrobacterium tumefaciens strain CP4 present in MON88017. MON832Monsanto Company Introduction, by particle bombardment, of glyphosateoxidase (GOX) and a modified 5- enolpyruvyl shikimate-3-phosphatesynthase (EPSPS), an enzyme involved in the shikimate biochemicalpathway for the production of the aromatic amino acids. MON863 MonsantoCompany Corn rootworm resistant maize produced by inserting the Cry3Bb1gene from Bacillus thuringiensis subsp. kumamotoensis. MON863 × MON810Monsanto Company Stacked insect resistant corn hybrid derived fromconventional cross-breeding of the parental lines MON863 (OECDidentifier: MON-OO863-5) and MON810 (OECD identifier: MON-OO81O-6)MON863 × M0N810 × Monsanto Company Stacked insect resistant andherbicide tolerant NK603 corn hybrid derived from conventional cross-breeding of the stacked hybrid MON-OO863-5 × MON-OO81O-6 and NK603 (OECDidentifier: MON-OO6O3-6). MON863 × NK603 Monsanto Company Stacked insectresistant and herbicide tolerant corn hybrid derived from conventionalcross- breeding of the parental lines MON863 (OECD identifier:MON-OO863-5) and NK603 (OECD identifier: MON-OO6O3-6). MON87460 MonsantoCompany MON 87460 was developed to provide reduced yield loss underwater-limited conditions compared to conventional maize. Efficacy in MON87460 is derived by expression of the inserted Bacillus subtilis coldshock protein B (CspB). MON88017 Monsanto Company Cornrootworm-resistant maize produced by inserting the Cry3Bb1 gene fromBacillus thuringiensis subspecies kumamotoensis strain EG4691.Glyphosate tolerance derived by inserting a5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene fromAgrobacterium tumefaciens strain CP4. MON89034 Monsanto Company Maizeevent expressing two different insecticidal proteins from Bacillusthuringiensis providing resistance to number of Lepidopteran pests.MON89034 × Monsanto Company Stacked insect resistant and glyphosatetolerant MON88017 maize derived from conventional cross-breeding of theparental lines MON89034 (OECD identifier: MON-89O34-3) and MON88017(OECD identifier: MON-88O17-3). Resistance to Lepidopteran insects isderived from two Cry genes present in MON89043. Corn rootworm resistanceis derived from a single Cry genes and glyphosate tolerance is derivedfrom the 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS) encodinggene from Agrobacterium tumefaciens present in MON88017. MON89034 ×NK603 Monsanto Company Stacked insect resistant and herbicide tolerantmaize produced by conventional cross breeding of parental lines MON89034(OECD identifier: MON- 89O34-3) with NK603 (OECD unique identifier:MON-OO6O3-6). Resistance to Lepidopteran insects is derived from two Crygenes present in MON89043. Tolerance to glyphosate herbicide is derivedfrom NK603. NK603 × MON810 Monsanto Company Stacked insect resistant andherbicide tolerant corn hybrid derived from conventional cross- breedingof the parental lines NK603 (OECD identifier: MON-OO6O3-6) and MON810(OECD identifier: MON-OO81O-6). MON89034 × TC1507 × Monsanto Company andStacked insect resistant and herbicide tolerant MON88017 × DAS- MycogenSeeds c/o Dow maize produced by conventional cross breeding of 59122-7AgroSciences LLC parental lines: MON89034, TC1507, MON88017, andDAS-59122. Resistance to the above-ground and below-ground insect pestsand tolerance to glyphosate and glufosinate-ammonium containingherbicides. MS3 Bayer CropScience Male sterility caused by expression ofthe barnase (Aventis ribonuclease gene from BacillusCropScience(AgrEvo)) amyloliquefaciens; PPT resistance was via PPT-acetyltransferase (PAT). MS6 Bayer CropScience Male sterility caused byexpression of the barnase (Aventis ribonuclease gene from BacillusCropScience(AgrEvo)) amyloliquefaciens; PPT resistance was via PPT-acetyltransferase (PAT). NK603 Monsanto Company Introduction, byparticle bombardment, of a modified 5-enolpyruvyl shikimate-3-phosphatesynthase (EPSPS), an enzyme involved in the shikimate biochemicalpathway for the production of the aromatic amino acids. NK603 × T25Monsanto Company Stacked glufosinate ammonium and glyphosate herbicidetolerant maize hybrid derived from conventional cross-breeding of theparental lines NK603 (OECD identifier: MON-OO6O3-6) and T25 (OECDidentifier: ACS-ZM003-2). T25 × MON810 Bayer CropScience Stacked insectresistant and herbicide tolerant (Aventis corn hybrid derived fromconventional cross- CropScience(AgrEvo)) breeding of the parental linesT25 (OECD identifier: ACS-ZMOO3-2) and MON810 (OECD identifier:MON-OO81O-6). TC1507 Mycogen (c/o Dow Insect-resistant and glufosinateammonium AgroSciences); Pioneer herbicide tolerant maize produced byinserting the (c/o DuPont) Cry1F gene from Bacillus thuringiensis var.aizawai and the phosphinothricin N- acetyltransferase encoding gene fromStreptomyces viridochromogenes. TC1507 × NK603 DOW AgroSciences LLCStacked insect resistant and herbicide tolerant corn hybrid derived fromconventional cross- breeding of the parental lines 1507 (OECDidentifier: DAS-O15O7-1) and NK603 (OECD identifier: MON-OO6O3-6).TC1507 × DAS-59122-7 DOW AgroSciences LLC Stacked insect resistant andherbicide tolerant and Pioneer Hi-Bred maize produced by conventionalcross breeding of International Inc. parental lines TC1507 (OECD uniqueidentifier: DAS-O15O7-1) with DAS-59122-7 (OECD unique identifier:DAS-59122-7). Resistance to Lepidopteran insects is derived from TC1507due the presence of the Cry1F gene from Bacillus thuringiensis var.aizawai. Corn rootworm- resistance is derived from DAS-59122-7 whichcontains the Cry34Ab1 and Cry35Ab1 genes from Bacillus thuringiensisstrain PS149B1. Tolerance to glufosinate ammonium herbicide is derivedfrom TC1507 from the phosphinothricin N- acetyltransferase encoding genefrom Streptomyces viridochromogenes.

Other events with regulatory approval are well known to one skilled inthe art and can be found at the Center for Environmental Risk Assessment(cera-gmc.org/?action=gm crop database, which can be accessed using thewww prefix) and at the International Service for the Acquisition ofAgri-Biotech Applications (isaaa.org/gmapprovaldatabase/default.asp,which can be accessed using the www prefix).

Gene Silencing

In some embodiments the stacked trait may be in the form of silencing ofone or more polynucleotides of interest resulting in suppression of oneor more target pest polypeptides. In some embodiments the silencing isachieved through the use of a suppression DNA construct.

In some embodiments one or more polynucleotide encoding the polypeptidesof the insecticidal polypeptides of the disclosure or fragments orvariants thereof may be stacked with one or more polynucleotidesencoding one or more polypeptides having insecticidal activity oragronomic traits as set forth supra and optionally may further includeone or more polynucleotides providing for gene silencing of one or moretarget polynucleotides as discussed infra.

“Suppression DNA construct” is a recombinant DNA construct which whentransformed or stably integrated into the genome of the plant, resultsin “silencing” of a target gene in the plant. The target gene may beendogenous or transgenic to the plant. “Silencing,” as used herein withrespect to the target gene, refers generally to the suppression oflevels of mRNA or protein/enzyme expressed by the target gene, and/orthe level of the enzyme activity or protein functionality. The term“suppression” includes lower, reduce, decline, decrease, inhibit,eliminate and prevent. “Silencing” or “gene silencing” does not specifymechanism and is inclusive, and not limited to, anti-sense,cosuppression, viral-suppression, hairpin suppression, stem-loopsuppression, RNAi-based approaches and small RNA-based approaches.

A suppression DNA construct may comprise a region derived from a targetgene of interest and may comprise all or part of the nucleic acidsequence of the sense strand (or antisense strand) of the target gene ofinterest. Depending upon the approach to be utilized, the region may be100% identical or less than 100% identical (e.g., at least 50% or anyinteger between 51% and 100% identical) to all or part of the sensestrand (or antisense strand) of the gene of interest.

Suppression DNA constructs are well-known in the art, are readilyconstructed once the target gene of interest is selected, and include,without limitation, cosuppression constructs, antisense constructs,viral-suppression constructs, hairpin suppression constructs, stem-loopsuppression constructs, double-stranded RNA-producing constructs, andmore generally, RNAi (RNA interference) constructs and small RNAconstructs such as siRNA (short interfering RNA) constructs and miRNA(microRNA) constructs.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target protein.

“Antisense RNA” refers to an RNA transcript that is complementary to allor part of a target primary transcript or mRNA and that blocks theexpression of a target isolated nucleic acid fragment (U.S. Pat. No.5,107,065). The complementarity of an antisense RNA may be with any partof the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, introns or the coding sequence.

“Cosuppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of the target protein. “Sense” RNArefers to RNA transcript that includes the mRNA and can be translatedinto protein within a cell or in vitro. Cosuppression constructs inplants have been previously designed by focusing on overexpression of anucleic acid sequence having homology to a native mRNA, in the senseorientation, which results in the reduction of all RNA having homologyto the overexpressed sequence (see, Vaucheret, et al., (1998) Plant J.16:651-659 and Gura, (2000) Nature 404:804-808).

Another variation describes the use of plant viral sequences to directthe suppression of proximal mRNA encoding sequences (PCT Publication WO1998/36083).

Recent work has described the use of “hairpin” structures thatincorporate all or part, of an mRNA encoding sequence in a complementaryorientation that results in a potential “stem-loop” structure for theexpressed RNA (PCT Publication WO 1999/53050). In this case the stem isformed by polynucleotides corresponding to the gene of interest insertedin either sense or anti-sense orientation with respect to the promoterand the loop is formed by some polynucleotides of the gene of interest,which do not have a complement in the construct. This increases thefrequency of cosuppression or silencing in the recovered transgenicplants. For review of hairpin suppression, see, Wesley, et al., (2003)Methods in Molecular Biology, Plant Functional Genomics: Methods andProtocols 236:273-286.

A construct where the stem is formed by at least 30 nucleotides from agene to be suppressed and the loop is formed by a random nucleotidesequence has also effectively been used for suppression (PCT PublicationWO 1999/61632).

The use of poly-T and poly-A sequences to generate the stem in thestem-loop structure has also been described (PCT Publication WO2002/00894).

Yet another variation includes using synthetic repeats to promoteformation of a stem in the stem-loop structure. Transgenic organismsprepared with such recombinant DNA fragments have been shown to havereduced levels of the protein encoded by the nucleotide fragment formingthe loop as described in PCT Publication WO 2002/00904.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire, et al., (1998) Nature 391:806). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing (PTGS) or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire, et al., (1999) TrendsGenet. 15:358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single-stranded RNA of viral genomicRNA. The presence of dsRNA in cells triggers the RNAi response through amechanism that has yet to be fully characterized.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein, et al., (2001) Nature 409:363).Short interfering RNAs derived from dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes (Elbashir, et al., (2001) Genes Dev. 15:188). Dicer has alsobeen implicated in the excision of 21- and 22-nucleotide small temporalRNAs (stRNAs) from precursor RNA of conserved structure that areimplicated in translational control (Hutvagner, et al., (2001) Science293:834). The RNAi response also features an endonuclease complex,commonly referred to as an RNA-induced silencing complex (RISC), whichmediates cleavage of single-stranded RNA having sequence complementarityto the antisense strand of the siRNA duplex. Cleavage of the target RNAtakes place in the middle of the region complementary to the antisensestrand of the siRNA duplex (Elbashir, et al., (2001) Genes Dev. 15:188).In addition, RNA interference can also involve small RNA (e.g., miRNA)mediated gene silencing, presumably through cellular mechanisms thatregulate chromatin structure and thereby prevent transcription of targetgene sequences (see, e.g., Allshire, (2002) Science 297:1818-1819;Volpe, et al., (2002) Science 297:1833-1837; Jenuwein, (2002) Science297:2215-2218 and Hall, et al., (2002) Science 297:2232-2237). As such,miRNA molecules of the disclosure can be used to mediate gene silencingvia interaction with RNA transcripts or alternately by interaction withparticular gene sequences, wherein such interaction results in genesilencing either at the transcriptional or post-transcriptional level.

Methods and compositions are further provided which allow for anincrease in RNAi produced from the silencing element. In suchembodiments, the methods and compositions employ a first polynucleotidecomprising a silencing element for a target pest sequence operablylinked to a promoter active in the plant cell; and, a secondpolynucleotide comprising a suppressor enhancer element comprising thetarget pest sequence or an active variant or fragment thereof operablylinked to a promoter active in the plant cell. The combined expressionof the silencing element with suppressor enhancer element leads to anincreased amplification of the inhibitory RNA produced from thesilencing element over that achievable with only the expression of thesilencing element alone. In addition to the increased amplification ofthe specific RNAi species itself, the methods and compositions furtherallow for the production of a diverse population of RNAi species thatcan enhance the effectiveness of disrupting target gene expression. Assuch, when the suppressor enhancer element is expressed in a plant cellin combination with the silencing element, the methods and compositioncan allow for the systemic production of RNAi throughout the plant; theproduction of greater amounts of RNAi than would be observed with justthe silencing element construct alone; and, the improved loading of RNAiinto the phloem of the plant, thus providing better control of phloemfeeding insects by an RNAi approach. Thus, the various methods andcompositions provide improved methods for the delivery of inhibitory RNAto the target organism. See, for example, US Patent ApplicationPublication 2009/0188008.

As used herein, a “suppressor enhancer element” comprises apolynucleotide comprising the target sequence to be suppressed or anactive fragment or variant thereof. It is recognize that the suppressorenhancer element need not be identical to the target sequence, butrather, the suppressor enhancer element can comprise a variant of thetarget sequence, so long as the suppressor enhancer element hassufficient sequence identity to the target sequence to allow for anincreased level of the RNAi produced by the silencing element over thatachievable with only the expression of the silencing element. Similarly,the suppressor enhancer element can comprise a fragment of the targetsequence, wherein the fragment is of sufficient length to allow for anincreased level of the RNAi produced by the silencing element over thatachievable with only the expression of the silencing element.

It is recognized that multiple suppressor enhancer elements from thesame target sequence or from different target sequences or fromdifferent regions of the same target sequence can be employed. Forexample, the suppressor enhancer elements employed can comprisefragments of the target sequence derived from different region of thetarget sequence (i.e., from the 3′UTR, coding sequence, intron, and/or5′UTR). Further, the suppressor enhancer element can be contained in anexpression cassette, as described elsewhere herein, and in specificembodiments, the suppressor enhancer element is on the same or on adifferent DNA vector or construct as the silencing element. Thesuppressor enhancer element can be operably linked to a promoter asdisclosed herein. It is recognized that the suppressor enhancer elementcan be expressed constitutively or alternatively, it may be produced ina stage-specific manner employing the various inducible ortissue-preferred or developmentally regulated promoters that arediscussed elsewhere herein.

In specific embodiments, employing both a silencing element and thesuppressor enhancer element the systemic production of RNAi occursthroughout the entire plant. In further embodiments, the plant or plantparts of the disclosure have an improved loading of RNAi into the phloemof the plant than would be observed with the expression of the silencingelement construct alone and, thus provide better control of phloemfeeding insects by an RNAi approach. In specific embodiments, theplants, plant parts and plant cells of the disclosure can further becharacterized as allowing for the production of a diversity of RNAispecies that can enhance the effectiveness of disrupting target geneexpression.

In specific embodiments, the combined expression of the silencingelement and the suppressor enhancer element increases the concentrationof the inhibitory RNA in the plant cell, plant, plant part, plant tissueor phloem over the level that is achieved when the silencing element isexpressed alone.

As used herein, an “increased level of inhibitory RNA” comprises anystatistically significant increase in the level of RNAi produced in aplant having the combined expression when compared to an appropriatecontrol plant. For example, an increase in the level of RNAi in theplant, plant part or the plant cell can comprise at least about a 1%,about a 1%-5%, about a 5%-10%, about a 10%-20%, about a 20%-30%, about a30%-40%, about a 40%-50%, about a 50%-60%, about 60-70%, about 70%-80%,about a 80%-90%, about a 90%-100% or greater increase in the level ofRNAi in the plant, plant part, plant cell or phloem when compared to anappropriate control. In other embodiments, the increase in the level ofRNAi in the plant, plant part, plant cell or phloem can comprise atleast about a 1 fold, about a 1 fold-5 fold, about a 5 fold-10 fold,about a 10 fold-20 fold, about a 20 fold-30 fold, about a 30 fold-40fold, about a 40 fold-50 fold, about a 50 fold-60 fold, about 60 fold-70fold, about 70 fold-80 fold, about a 80 fold-90 fold, about a 90fold-100 fold or greater increase in the level of RNAi in the plant,plant part, plant cell or phloem when compared to an appropriatecontrol. Examples of combined expression of the silencing element withsuppressor enhancer element for the control of Stinkbugs and Lygus canbe found in US Patent Application Publication 2011/0301223 and US PatentApplication Publication 2009/0192117.

Some embodiments relate to down-regulation of expression of target genesin insect pest species by interfering ribonucleic acid (RNA) molecules.PCT Publication WO 2007/074405 describes methods of inhibitingexpression of target genes in invertebrate pests including Coloradopotato beetle. PCT Publication WO 2005/110068 describes methods ofinhibiting expression of target genes in invertebrate pests including inparticular Western corn rootworm as a means to control insectinfestation. Furthermore, PCT Publication WO 2009/091864 describescompositions and methods for the suppression of target genes from insectpest species including pests from the Lygus genus. Nucleic acidmolecules including RNAi for targeting the vacuolar ATPase H subunit,useful for controlling a coleopteran pest population and infestation asdescribed in US Patent Application Publication 2012/0198586. PCTPublication WO 2012/055982 describes ribonucleic acid (RNA or doublestranded RNA) that inhibits or down regulates the expression of a targetgene that encodes: an insect ribosomal protein such as the ribosomalprotein L19, the ribosomal protein L40 or the ribosomal protein S27A; aninsect proteasome subunit such as the Rpn6 protein, the Pros 25, theRpn2 protein, the proteasome beta 1 subunit protein or the Pros beta 2protein; an insect β-coatomer of the COPI vesicle, the y-coatomer of theCOPI vesicle, the β′-coatomer protein or the ζ-coatomer of the COPIvesicle; an insect Tetraspanine 2 A protein which is a putativetransmembrane domain protein; an insect protein belonging to the actinfamily such as Actin 5C; an insect ubiquitin-5E protein; an insect Sec23protein which is a GTPase activator involved in intracellular proteintransport; an insect crinkled protein which is an unconventional myosinwhich is involved in motor activity; an insect crooked neck proteinwhich is involved in the regulation of nuclear alternative mRNAsplicing; an insect vacuolar H+-ATPase G-subunit protein and an insectTbp-1 such as Tat-binding protein. US Patent Application Publications2012/029750, US 20120297501, and 2012/0322660 describe interferingribonucleic acids (RNA or double stranded RNA) that functions uponuptake by an insect pest species to down-regulate expression of a targetgene in said insect pest, wherein the RNA comprises at least onesilencing element wherein the silencing element is a region ofdouble-stranded RNA comprising annealed complementary strands, onestrand of which comprises or consists of a sequence of nucleotides whichis at least partially complementary to a target nucleotide sequencewithin the target gene. US Patent Application Publication 2012/0164205describe potential targets for interfering double stranded ribonucleicacids for inhibiting invertebrate pests including: a Chd3 HomologousSequence, a Beta-Tubulin Homologous Sequence, a 40 kDa V-ATPaseHomologous Sequence, a EF1α Homologous Sequence, a 26S ProteosomeSubunit p28 Homologous Sequence, a Juvenile Hormone Epoxide HydrolaseHomologous Sequence, a Swelling Dependent Chloride Channel ProteinHomologous Sequence, a Glucose-6-Phosphate 1-Dehydrogenase ProteinHomologous Sequence, an Act42A Protein Homologous Sequence, aADP-Ribosylation Factor 1 Homologous Sequence, a Transcription FactorIIB Protein Homologous Sequence, a Chitinase Homologous Sequences, aUbiquitin Conjugating Enzyme Homologous Sequence, aGlyceraldehyde-3-Phosphate Dehydrogenase Homologous Sequence, anUbiquitin B Homologous Sequence, a Juvenile Hormone Esterase Homolog,and an Alpha Tubuliln Homologous Sequence.

Use in Pesticidal Control

General methods for employing strains comprising a nucleic acid sequenceof the embodiments or a variant thereof, in pesticide control or inengineering other organisms as pesticidal agents are known in the art.See, for example U.S. Pat. No. 5,039,523 and EP 0480762A2.

Microorganism hosts that are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one ormore crops of interest may be selected. These microorganisms areselected so as to be capable of successfully competing in the particularenvironment with the wild-type microorganisms, provide for stablemaintenance and expression of the gene expressing the insecticidalpolypeptide of the disclosure, and desirably, provide for improvedprotection of the pesticide from environmental degradation andinactivation.

Such microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms such as bacteria, e.g., Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus,Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes, fungi,particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces,Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interestare such phytosphere bacterial species as Pseudomonas syringae,Pseudomonas fluorescens, Pseudomonas chlororaphis, Serratia marcescens,Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides,Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus,Clavibacter xyli and Azotobacter vinelandii and phytosphere yeastspecies such as Rhodotorula rubra, R. glutinis, R. marina, R.aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii,Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomycesroseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans.Of particular interest are the pigmented microorganisms. Host organismsof particular interest include yeast, such as Rhodotorula spp.,Aureobasidium spp., Saccharomyces spp. (such as S. cerevisiae),Sporobolomyces spp., phylloplane organisms such as Pseudomonas spp.(such as P. aeruginosa, P. fluorescens, P. chlororaphis), Erwinia spp.,and Flavobacterium spp., and other such organisms, includingAgrobacterium tumefaciens, E. coli, Bacillus subtilis, Bacillus cereusand the like.

Genes encoding the insecticidal polypeptides of the embodiments can beintroduced into microorganisms that multiply on plants (epiphytes) todeliver insecticidal polypeptides to potential target pests. Epiphytes,for example, can be gram-positive or gram-negative bacteria.

Root-colonizing bacteria, for example, can be isolated from the plant ofinterest by methods known in the art. Specifically, a Bacillus cereusstrain that colonizes roots can be isolated from roots of a plant (see,for example, Handelsman et al. (1991) Appl. Environ. Microbiol.56:713-718). Genes encoding the insecticidal polypeptides of theembodiments can be introduced into a root-colonizing Bacillus cereus bystandard methods known in the art.

Genes encoding insecticidal polypeptides of the disclosure can beintroduced, for example, into the root-colonizing Bacillus by means ofelectro transformation. Specifically, genes encoding the insecticidalpolypeptides of the disclosure can be cloned into a shuttle vector, forexample, pHT3101 (Lerecius, et al., (1989) FEMS Microbiol. Letts.60:211-218. The shuttle vector pHT3101 containing the coding sequencefor the particular insecticidal polypeptide gene of the disclosure can,for example, be transformed into the root-colonizing Bacillus by meansof electroporation (Lerecius, et al., (1989) FEMS Microbiol. Letts.60:211-218).

Expression systems can be designed so that insecticidal polypeptides ofthe disclosure are secreted outside the cytoplasm of gram-negativebacteria, such as E. coli, for example. Advantages of havinginsecticidal polypeptides of the disclosure secreted are: (1) avoidanceof potential cytotoxic effects of the insecticidal polypeptide of thedisclosure expressed; and (2) improvement in the efficiency ofpurification of the insecticidal polypeptide of the disclosure,including, but not limited to, increased efficiency in the recovery andpurification of the protein per volume cell broth and decreased timeand/or costs of recovery and purification per unit protein.

Insecticidal polypeptides of the disclosure can be made to be secretedin E. coli, for example, by fusing an appropriate E. coli signal peptideto the amino-terminal end of the insecticidal polypeptide of thedisclosure. Signal peptides recognized by E. coli can be found inproteins already known to be secreted in E. coli, for example the OmpAprotein (Ghrayeb, et al., (1984) EMBO J, 3:2437-2442). OmpA is a majorprotein of the E. coli outer membrane, and thus its signal peptide isthought to be efficient in the translocation process. Also, the OmpAsignal peptide does not need to be modified before processing as may bethe case for other signal peptides, for example lipoprotein signalpeptide (Duffaud, et al., (1987) Meth. Enzymol. 153:492).

Insecticidal polypeptides of the embodiments can be fermented in abacterial host and the resulting bacteria processed and used as amicrobial spray in the same manner that Bt strains have been used asinsecticidal sprays. In the case of an insecticidal polypeptide of thedisclosure(s) that is secreted from Bacillus, the secretion signal isremoved or mutated using procedures known in the art. Such mutationsand/or deletions prevent secretion of the insecticidal polypeptide(s)into the growth medium during the fermentation process. The insecticidalpolypeptides of the disclosure are retained within the cell, and thecells are then processed to yield the encapsulated insecticidalpolypeptides. Any suitable microorganism can be used for this purpose.Pseudomonas has been used to express Bt toxins as encapsulated proteinsand the resulting cells processed and sprayed as an insecticide(Gaertner, et al., (1993), in: Advanced Engineered Pesticides, ed. Kim).

Alternatively, the insecticidal polypeptides of the disclosure areproduced by introducing a heterologous gene into a cellular host.Expression of the heterologous gene results, directly or indirectly, inthe intracellular production and maintenance of the pesticide. Thesecells are then treated under conditions that prolong the activity of thetoxin produced in the cell when the cell is applied to the environmentof target pest(s). The resulting product retains the toxicity of thetoxin. These naturally encapsulated insecticidal polypeptides may thenbe formulated in accordance with conventional techniques for applicationto the environment hosting a target pest, e.g., soil, water, and foliageof plants. See, for example EPA 0192319, and the references citedtherein.

Pesticidal Compositions

In some embodiments the active ingredients can be applied in the form ofcompositions and can be applied to the crop area or plant to be treated,simultaneously or in succession, with other compounds. These compoundscan be fertilizers, weed killers, Cryoprotectants, surfactants,detergents, pesticidal soaps, dormant oils, polymers, and/ortime-release or biodegradable carrier formulations that permit long-termdosing of a target area following a single application of theformulation. They can also be selective herbicides, chemicalinsecticides, virucides, microbicides, amoebicides, pesticides,fungicides, bacteriocides, nematocides, molluscicides or mixtures ofseveral of these preparations, if desired, together with furtheragriculturally acceptable carriers, surfactants or application-promotingadjuvants customarily employed in the art of formulation. Suitablecarriers and adjuvants can be solid or liquid and correspond to thesubstances ordinarily employed in formulation technology, e.g. naturalor regenerated mineral substances, solvents, dispersants, wettingagents, tackifiers, binders or fertilizers. Likewise the formulationsmay be prepared into edible “baits” or fashioned into pest “traps” topermit feeding or ingestion by a target pest of the pesticidalformulation.

Methods of applying an active ingredient or an agrochemical compositionthat contains at least one of the insecticidal polypeptides of thedisclosure produced by the bacterial strains include leaf application,seed coating and soil application. The number of applications and therate of application depend on the intensity of infestation by thecorresponding pest.

The composition may be formulated as a powder, dust, pellet, granule,spray, emulsion, colloid, solution or such like, and may be prepared bysuch conventional means as desiccation, lyophilization, homogenation,extraction, filtration, centrifugation, sedimentation or concentrationof a culture of cells comprising the polypeptide. In all suchcompositions that contain at least one such pesticidal polypeptide, thepolypeptide may be present in a concentration of from about 1% to about99% by weight.

Lepidopteran, Dipteran, Heteropteran, nematode, Hemiptera or Coleopteranpests may be killed or reduced in numbers in a given area by the methodsof the disclosure or may be prophylactically applied to an environmentalarea to prevent infestation by a susceptible pest. Preferably the pestingests or is contacted with, a pesticidally-effective amount of thepolypeptide. “Pesticidally-effective amount” as used herein refers to anamount of the pesticide that is able to bring about death to at leastone pest or to noticeably reduce pest growth, feeding or normalphysiological development. This amount will vary depending on suchfactors as, for example, the specific target pests to be controlled, thespecific environment, location, plant, crop or agricultural site to betreated, the environmental conditions and the method, rate,concentration, stability, and quantity of application of thepesticidally-effective polypeptide composition. The formulations mayalso vary with respect to climatic conditions, environmentalconsiderations, and/or frequency of application and/or severity of pestinfestation.

The pesticide compositions described may be made by formulating eitherthe bacterial cell, Crystal and/or spore suspension or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, desiccated or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial or a suspension in oil (vegetable or mineral) or water oroil/water emulsions or as a wettable powder or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,inert components, dispersants, surfactants, tackifiers, binders, etc.that are ordinarily used in pesticide formulation technology; these arewell known to those skilled in pesticide formulation. The formulationsmay be mixed with one or more solid or liquid adjuvants and prepared byvarious means, e.g., by homogeneously mixing, blending and/or grindingthe pesticidal composition with suitable adjuvants using conventionalformulation techniques. Suitable formulations and application methodsare described in U.S. Pat. No. 6,468,523, herein incorporated byreference. The plants can also be treated with one or more chemicalcompositions, including one or more herbicide, insecticides orfungicides. Exemplary chemical compositions include: Fruits/VegetablesHerbicides: Atrazine, Bromacil, Diuron, Glyphosate, Linuron, Metribuzin,Simazine, Trifluralin, Fluazifop, Glufosinate, Halo sulfuron Gowan,Paraquat, Propyzamide, Sethoxydim, Butafenacil, Halosulfuron,Indaziflam; Fruits/Vegetables Insecticides: Aldicarb, Bacillusthuriengiensis, Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin,Deltamethrin, Diazinon, Malathion, Abamectin,Cyfluthrin/beta-cyfluthrin, Esfenvalerate, Lambda-cyhalothrin,Acequinocyl, Bifenazate, Methoxyfenozide, Novaluron, Chromafenozide,Thiacloprid, Dinotefuran, FluaCrypyrim, Tolfenpyrad, Clothianidin,Spirodiclofen, Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr,Cyazypyr, Spinoteram, Triflumuron, Spirotetramat, Imidacloprid,Flubendiamide, Thiodicarb, Metaflumizone, Su lfoxaflor, Cyflumetofen,Cyanopyrafen, Imidacloprid, Clothianidin, Thiamethoxam, Spinotoram,Thiodicarb, Flonicamid, Methiocarb, Emamectin-benzoate, lndoxacarb,Forthiazate, Fenamiphos, Cadusaphos, Pyriproxifen, Fenbutatin-oxid,Hexthiazox, Methomyl,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on;Fruits/Vegetables Fungicides: Carbendazim, Chlorothalonil, EBDCs,Sulphur, Thiophanate-methyl, Azoxystrobin, Cymoxanil, Fluazinam,Fosetyl, Iprodione, Kresoxim-methyl, Metalaxyl/mefenoxam,Trifloxystrobin, Ethaboxam, 1provalicarb, Trifloxystrobin, Fenhexamid,Oxpoconazole fumarate, Cyazofamid, Fenamidone, Zoxamide, Picoxystrobin,Pyraclostrobin, Cyflufenamid, Boscalid; Cereals Herbicides: Isoproturon,Bromoxynil, loxynil, Phenoxies, Chlorsulfuron, Clodinafop, Diclofop,Diflufenican, Fenoxaprop, Florasulam, Fluoroxypyr, Metsulfuron,Triasulfuron, Flucarbazone, lodosulfuron, Propoxycarbazone, Picolinafen,Mesosulfuron, Beflubutamid, Pinoxaden, Amidosulfuron, ThifensulfuronMethyl, Tribenuron, Flupyrsulfuron, Sulfosulfuron, Pyrasulfotole,Pyroxsulam, Flufenacet, Tralkoxydim, Pyroxasulfon; Cereals Fungicides:Carbendazim, Chlorothalonil, Azoxystrobin, Cyproconazole, Cyprodinil,Fenpropimorph, Epoxiconazole, Kresoxim-methyl, Quinoxyfen, Tebuconazole,Trifloxystrobin, Simeconazole, Picoxystrobin, Pyraclostrobin,Dimoxystrobin, Prothioconazole, Fluoxastrobin; Cereals Insecticides:Dimethoate, Lambda-cyhalthrin, Deltamethrin, alpha-Cypermethrin,β-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Clorphyriphos, Metamidophos,Oxidemethon-methyl, Pirimicarb, Methiocarb; Maize Herbicides: Atrazine,Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, (S-)Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor,Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione,Foramsulfuron, Topramezone, Tembotrione, Saflufenacil, Thiencarbazone,Flufenacet, Pyroxasulfon; Maize Insecticides: Carbofuran, Chlorpyrifos,Bifenthrin, Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin,Terbufos, Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide,Triflumuron, Rynaxypyr, Deltamethrin, Thiodicarb, β-Cyfluthrin,Cypermethrin, Bifenthrin, Lufenuron, Triflumoron,Tefluthrin,Tebupirimphos, Ethiprole, Cyazypyr, Thiacloprid, Acetamiprid,Dinetofuran, Avermectin, Methiocarb, Spirodiclofen, Spirotetramat; MaizeFungicides: Fenitropan, Thiram, Prothioconazole, Tebuconazole,Trifloxystrobin; Rice Herbicides: Butachlor, Propanil, Azimsulfuron,Bensulfuron, Cyhalofop, Daimuron, Fentrazamide, Imazosulfuron,Mefenacet, Oxaziclomefone, Pyrazosulfuron, Pyributicarb, Quinclorac,Thiobencarb, Indanofan, Flufenacet, Fentrazamide, Halosulfuron,Oxaziclomefone, Benzobicyclon, Pyriftalid, Penoxsulam, Bispyribac,Oxadiargyl, Ethoxysulfuron, Pretilachlor, Mesotrione, Tefuryltrione,Oxadiazone, Fenoxaprop, Pyrimisulfan; Rice Insecticides: Diazinon,Fenitrothion, Fenobucarb, Monocrotophos, Benfuracarb, Buprofezin,Dinotefuran, Fipronil, Imidacloprid, Isoprocarb, Thiacloprid,Chromafenozide, Thiacloprid, Dinotefuran, Clothianidin, Ethiprole,Flubendiamide, Rynaxypyr, Deltamethrin, Acetamiprid, Thiamethoxam,Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Cypermethrin,Chlorpyriphos, Cartap, Methamidophos, Etofenprox, Triazophos,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Carbofuran, Benfuracarb; Rice Fungicides: Thiophanate-methyl,Azoxystrobin, Carpropamid, Edifenphos, Ferimzone, Iprobenfos,Isoprothiolane, Pencycuron, Probenazole, Pyroquilon, Tricyclazole,Trifloxystrobin, Diclocymet, Fenoxanil, Simeconazole, Tiadinil; CottonHerbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn,Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate,Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron,Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; CottonInsecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin,Deltamethrin, Malathion, Monocrotophos, Abamectin, Acetamiprid,Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin,Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl,Flonicamid, Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin,Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran,Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin,4-[[(6-Chlorpyridin-3-Amethyl](2,2-difluorethyl)amino]furan-2(5H)-on,Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen,Sulfoxaflor, Profenophos, Thriazophos, Endosulfan; Cotton Fungicides:Etridiazole, Metalaxyl, Quintozene; Soybean Herbicides: Alachlor,Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl,Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin,Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim,Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl,Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr,Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole,Deltamethrin, ϵ-Cyfluthrin, gamma and lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-Amethyl](2,2-difluorethyl)amino]furan-2(5H)-on,Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb,beta-Cyfluthrin; Soybean Fungicides: Azoxystrobin, Cyproconazole,Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole,Trifloxystrobin, Prothioconazole, Tetraconazole; Sugarbeet Herbicides:Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate,Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim,Triflusulfuron, Tepraloxydim, Quizalofop; Sugarbeet Insecticides:Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid,Dinetofuran, Deltamethrin, β-Cyfluthrin, gamma/lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran; CanolaHerbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate,Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop,Clethodim, Tepraloxydim; Canola Fungicides: Azoxystrobin, Carbendazim,Fludioxonil, Iprodione, Prochloraz, Vinclozolin; Canola Insecticides:Carbofuran organophosphates, Pyrethroids, Thiacloprid, Deltamethrin,Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofuran,β-Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole,Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on.

In some embodiments the herbicide is Atrazine, Bromacil, Diuron,Chlorsulfuron, Metsulfuron, Thifensulfuron Methyl, Tribenuron,Acetochlor, Dicamba, Isoxaflutole, Nicosulfuron, Rimsulfuron,Pyrithiobac-sodium, Flumioxazin, Chlorimuron-Ethyl, Metribuzin,Quizalofop, S-metolachlor, Hexazinne or combinations thereof.

In some embodiments the insecticide is Esfenvalerate,Chlorantraniliprole, Methomyl, lndoxacarb, Oxamyl or combinationsthereof.

Pesticidal and Insecticidal Activity

“Pest” includes but is not limited to, insects, fungi, bacteria,nematodes, mites, ticks and the like. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyLepidoptera and Coleoptera.

Those skilled in the art will recognize that not all compounds areequally effective against all pests. Compounds of the embodimentsdisplay activity against insect pests, which may include economicallyimportant agronomic, forest, greenhouse, nursery ornamentals, food andfiber, public and animal health, domestic and commercial structure,household and stored product pests.

Larvae of the order Lepidoptera include, but are not limited to,armyworms, cutworms, loopers and heliothines in the family NoctuidaeSpodoptera frugiperda J E Smith (fall armyworm); S. exigua Hübner (beetarmyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar);Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus(cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogoniaMorrison (western cutworm); A. subterranea Fabricius (granulatecutworm); Alabama argillacea Hübner (cotton leaf worm); Trichoplusia niHübner (cabbage looper); Pseudoplusia includens Walker (soybean looper);Anticarsia gemmatalis Hübner (velvetbean caterpillar); Hypena scabraFabricius (green cloverworm); Heliothis virescens Fabricius (tobaccobudworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindaraBarnes and Mcdunnough (rough skinned cutworm); Athetis lepigone; Euxoamessoria Harris (darksided cutworm); Earias insulana Boisduval (spinybollworm); E. vittella Fabricius (spotted bollworm); Helicoverpaarmigera Hübner (American bollworm); H. zea Boddie (corn earworm orcotton bollworm); Melanchra picta Harris (zebra caterpillar); Egira(Xylomyges) curialis Grote (citrus cutworm); borers, casebearers,webworms, coneworms; Sesamia inferens (Asiatic pink stem borer), andskeletonizers from the family Pyralidae Ostrinia nubilalis Hübner(European corn borer); Amyelois transitella Walker (naval orangeworm);Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautellaWalker (almond moth); Chilo suppressalis Walker (rice stem borer); C.partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth);Crambus caliginosellus Clemens (corn root webworm); C. teterrellusZincken (bluegrass webworm); Cnaphalocrocis medinalis Guenée (rice leafroller); Desmia funeralis Hübner (grape leaffolder); Diaphania hyalinataLinnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraeagrandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius(surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestiaelutella Hübner (tobacco (cacao) moth); Galleria mellonella Linnaeus(greater wax moth); Herpetogramma licarsisalis Walker (sod webworm);Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellusZeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser waxmoth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalisWalker (tea tree web moth); Maruca testulalis Geyer (bean pod borer);Plodia interpunctella Hübner (Indian meal moth); Scirpophaga incertulasWalker (yellow stem borer); Udea rubigalis Guenée (celery leaftier); andleafrollers, budworms, seed worms and fruit worms in the familyTortricidae Acleris gloverana Walsingham (Western blackheaded budworm);A. variana Fernald (Eastern blackheaded budworm); Archips argyrospilaWalker (fruit tree leaf roller); A. rosana Linnaeus (European leafroller); and other Archips species, Adoxophyes orana Fischer vonRosslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham(banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C.pomonella Linnaeus (coding moth); Platynota flavedana Clemens(variegated leafroller); P. stultana Walsingham (omnivorous leafroller);Lobesia botrana Denis & Schiffermuller (European grape vine moth);Spilonota ocellana Denis & Schiffermuller (eyespotted bud moth);Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguellaHübner (vine moth); Bonagota salubricola Meyrick (Brazilian appleleafroller); Grapholita molesta Busck (oriental fruit moth); Suleimahelianthana Riley (sunflower bud moth); Argyrotaenia spp.; Choristoneuraspp.

Selected other agronomic pests in the order Lepidoptera include, but arenot limited to, Alsophila pometaria Harris (fall cankerworm); Anarsialineatella Zeller (peach twig borer); Anisota senatoria J. E. Smith(orange striped oakworm); Antheraea pernyi Gurin-Meneville (Chinese OakTussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiellaBusck (cotton leaf perforator); Collas eurytheme Boisduval (alfalfacaterpillar); Conogethes punctiferalis (Yellow Peach Moth); Datanaintegerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricusTschetwerikov (Siberian silk moth), Ennomos subsignaria Hübner (elmspanworm); Erannis tiliaria Harris (linden looper); Euproctischrysorrhoea Linnaeus (browntail moth); Harrisina americanaGurin-Meneville (grapeleaf skeletonizer); Hemileuca oliviae Cockrell(range caterpillar); Hyphantria cunea Drury (fall webworm); Keiferialycopersicella Walsingham (tomato pinworm); Lambdina fiscellariafiscellaria Hulst (Eastern hemlock looper); L. fiscellaria lugubrosaHulst (Western hemlock looper); Leucoma salicis Linnaeus (satin moth);Lymantria dispar Linnaeus (gypsy moth); Manduca quinquemaculata Haworth(five spotted hawk moth, tomato hornworm); M. sexta Haworth (tomatohornworm, tobacco hornworm); Operophtera brumata Linnaeus (winter moth);Paleacrita vernata Peck (spring cankerworm); Papilio cresphontes Cramer(giant swallowtail orange dog); Phryganidia califomica Packard(California oakworm); Phyllocnistis citrella Stainton (citrusleafminer); Phyllonorycter blancardella Fabricius (spotted tentiformleafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapaeLinnaeus (small white butterfly); P. napi Linnaeus (green veined whitebutterfly); Platyptilia carduidactyla Riley (artichoke plume moth);Plutella xylostella Linnaeus (diamondback moth); Pectinophoragossypiella Saunders (pink bollworm); Pontia protodice Boisduval andLeconte (Southern cabbageworm); Sabulodes aegrotata Guenée (omnivorouslooper); Schizura concinna J. E. Smith (red humped caterpillar);Sitotroga cerealella Olivier (Angoumois grain moth); Thaumetopoeapityocampa Schiffermuller (pine processionary caterpillar); Tineolabisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick (tomatoleafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothissubflexa Guenée; Malacosoma spp. and Orgyia spp.

Of interest are larvae and adults of the order Coleoptera includingweevils from the families Anthribidae, Bruchidae and Curculionidae(including, but not limited to: Anthonomus grandis Boheman (bollweevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil);Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (riceweevil); Hypera punctata Fabricius (clover leaf weevil);Cylindrocopturus adspersus LeConte (sunflower stem weevil); Smicronyxfulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (graysunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug));flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetlesand leafminers in the family Chrysomelidae (including, but not limitedto: Leptinotarsa decemlineata Say (Colorado potato beetle); Diabroticavirgifera virgifera LeConte (western corn rootworm); D. barberi Smithand Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber(southern corn rootworm); Chaetocnema pulicaria Melsheimer (corn fleabeetle); Phyllotreta cruciferae Goeze (Crucifer flea beetle);Phyllotreta striolata (stripped flea beetle); Colaspis brunnea Fabricius(grape colaspis); Oulema melanopus Linnaeus (cereal leaf beetle);Zygogramma exclamationis Fabricius (sunflower beetle)); beetles from thefamily Coccinellidae (including, but not limited to: Epilachnavarivestis Mulsant (Mexican bean beetle)); chafers and other beetlesfrom the family Scarabaeidae (including, but not limited to: Popilliajaponica Newman (Japanese beetle); Cyclocephala borealis Arrow (northernmasked chafer, white grub); C. immaculata Olivier (southern maskedchafer, white grub); Rhizotrogus majalis Razoumowsky (European chafer);Phyllophaga crinita Burmeister (white grub); Ligyrus gibbosus De Geer(carrot beetle)); carpet beetles from the family Dermestidae; wirewormsfrom the family Elateridae, Eleodes spp., Melanotus spp.; Conoderusspp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; barkbeetles from the family Scolytidae and beetles from the familyTenebrionidae.

Adults and immatures of the order Diptera are of interest, includingleafminers Agromyza parvicornis Loew (corn blotch leafminer); midges(including, but not limited to: Contarinia sorghicola Coquillett(sorghum midge); Mayetiola destructor Say (Hessian fly); Sitodiplosismosellana Gehin (wheat midge); Neolasioptera murtfeldtiana Felt,(sunflower seed midge)); fruit flies (Tephritidae), Oscinella fritLinnaeus (fruit flies); maggots (including, but not limited to: Deliaplatura Meigen (seedcorn maggot); D. coarctata Fallen (wheat bulb fly)and other Delia spp., Meromyza americana Fitch (wheat stem maggot);Musca domestica Linnaeus (house flies); Fannia canicularis Linnaeus, F.femoralis Stein (lesser house flies); Stomoxys calcitrans Linnaeus(stable flies)); face flies, horn flies, blow flies, Chrysomya spp.;Phormia spp. and other muscoid fly pests, horse flies Tabanus spp.; botflies Gastrophilus spp.; Oestrus spp.; cattle grubs Hypoderma spp.; deerflies Chrysops spp.; Melophagus ovinus Linnaeus (keds) and otherBrachycera, mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; blackflies Prosimulium spp.; Simulium spp.; biting midges, sand flies,sciarids, and other Nematocera.

Included as insects of interest are adults and nymphs of the ordersHemiptera and Homoptera such as, but not limited to, adelgids from thefamily Adelgidae, plant bugs from the family Miridae, cicadas from thefamily Cicadidae, leafhoppers, Empoasca spp.; from the familyCicadellidae, planthoppers from the families Cixiidae, Flatidae,Fulgoroidea, Issidae and Delphacidae, treehoppers from the familyMembracidae, psyllids from the family Psyllidae, whiteflies from thefamily Aleyrodidae, aphids from the family Aphididae, phylloxera fromthe family Phylloxeridae, mealybugs from the family Pseudococcidae,scales from the families Asterolecanidae, Coccidae, Dactylopiidae,Diaspididae, Eriococcidae Ortheziidae, Phoenicococcidae andMargarodidae, lace bugs from the family Tingidae, stink bugs from thefamily Pentatomidae, cinch bugs, Blissus spp.; and other seed bugs fromthe family Lygaeidae, spittlebugs from the family Cercopidae squash bugsfrom the family Coreidae and red bugs and cotton stainers from thefamily Pyrrhocoridae.

Agronomically important members from the order Homoptera furtherinclude, but are not limited to: Acyrthisiphon pisum Harris (pea aphid);Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black beanaphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicisForbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecolaPatch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid);Chaetosiphon fragaefolii Cockerell (strawberry aphid); Diuraphis noxiaKurdjumov/Mordvilko (Russian wheat aphid); Dysaphis plantagineaPaaserini (rosy apple aphid); Eriosoma lanigerum Hausmann (woolly appleaphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopteruspruni Geoffroy (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnipaphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphumeuphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach-potatoaphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid);Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch(corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Schizaphisgraminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcaneaphid); Sitobion avenae Fabricius (English grain aphid); Therioaphismaculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer deFonscolombe (black citrus aphid) and T. citricida Kirkaldy (brown citrusaphid); Adelges spp. (adelgids); Phylloxera devastatrix Pergande (pecanphylloxera); Bemisia tabaci Gennadius (tobacco whitefly, sweetpotatowhitefly); B. argentifolii Bellows & Perring (silverleaf whitefly);Dialeurodes citri Ashmead (citrus whitefly); Trialeurodes abutiloneus(bandedwinged whitefly) and T. vaporariorum Westwood (greenhousewhitefly); Empoasca fabae Harris (potato leafhopper); Laodelphaxstriatellus Fallen (smaller brown planthopper); Macrolestesquadrilineatus Forbes (aster leafhopper); Nephotettix cinticeps Uhler(green leafhopper); N. nigropictus Stal (rice leafhopper); Nilaparvatalugens Stal (brown planthopper); Peregrinus maidis Ashmead (cornplanthopper); Sogatella furcifera Horvath (white-backed planthopper);Sogatodes orizicola Muir (rice delphacid); Typhlocyba pomaria McAtee(white apple leafhopper); Erythroneoura spp. (grape leafhoppers);Magicicada septendecim Linnaeus (periodical cicada); Icerya purchasiMaskell (cottony cushion scale); Quadraspidiotus perniciosus Comstock(San Jose scale); Planococcus citri Risso (citrus mealybug);Pseudococcus spp. (other mealybug complex); Cacopsylla pyricola Foerster(pear psylla); Trioza diospyri Ashmead (persimmon psylla).

Agronomically important species of interest from the order Hemipterainclude, but are not limited to: Acrosternum hilare Say (green stinkbug); Anasa tristis De Geer (squash bug); Blissus leucopterusleucopterus Say (chinch bug); Corythuca gossypii Fabricius (cotton lacebug); Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellusHerrich-Schaffer (cotton stainer); Euschistus servus Say (brown stinkbug); E. variolarius Palisot de Beauvois (one-spotted stink bug);Graptostethus spp. (complex of seed bugs); Leptoglossus corculus Say(leaf-footed pine seed bug); Lygus lineolaris Palisot de Beauvois(tarnished plant bug); L. hesperus Knight (Western tarnished plant bug);L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius(European tarnished plant bug); Lygocoris pabulinus Linnaeus (commongreen capsid); Nezara viridula Linnaeus (southern green stink bug);Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas(large milkweed bug); Pseudatomoscelis seriatus Reuter (cottonfleahopper).

Furthermore, embodiments may be effective against Hemiptera such,Calocoris norvegicus Gmelin (strawberry bug); Orthops campestrisLinnaeus; Plesiocoris rugicollis Fallen (apple capsid); Cyrtopeltismodestus Distant (tomato bug); Cyrtopeltis notatus Distant (suckfly);Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Diaphnocorischlorionis Say (honeylocust plant bug); Labopidicola allii Knight (onionplant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper);Adelphocoris rapidus Say (rapid plant bug); Poecilocapsus lineatusFabricius (four-lined plant bug); Nysius ericae Schilling (false chinchbug); Nysius raphanus Howard (false chinch bug); Nezara viridulaLinnaeus (Southern green stink bug); Eurygaster spp.; Coreidae spp.;Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Reduviidae spp.and Cimicidae spp.

Also included are adults and larvae of the order Acari (mites) such asAceria tosichella Keifer (wheat curl mite); Petrobia latens Muller(brown wheat mite); spider mites and red mites in the familyTetranychidae, Panonychus ulmi Koch (European red mite); Tetranychusurticae Koch (two spotted spider mite); (T. mcdanieli McGregor (McDanielmite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestaniUgarov & Nikolski (strawberry spider mite); flat mites in the familyTenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust andbud mites in the family Eriophyidae and other foliar feeding mites andmites important in human and animal health, i.e., dust mites in thefamily Epidermoptidae, follicle mites in the family Demodicidae, grainmites in the family Glycyphagidae, ticks in the order Ixodidae. Ixodesscapularis Say (deer tick); I. holocyclus Neumann (Australian paralysistick); Dermacentor variabilis Say (American dog tick); Amblyommaamericanum Linnaeus (lone star tick) and scab and itch mites in thefamilies Psoroptidae, Pyemotidae and Sarcoptidae.

Insect pests of the order Thysanura are of interest, such as Lepismasaccharina Linnaeus (silverfish); Thermobia domestica Packard(firebrat).

Additional arthropod pests covered include: spiders in the order Araneaesuch as Loxosceles reclusa Gertsch and Mulaik (brown recluse spider) andthe Latrodectus mactans Fabricius (black widow spider) and centipedes inthe order Scutigeromorpha such as Scutigera coleoptrata Linnaeus (housecentipede).

Insect pest of interest include the superfamily of stink bugs and otherrelated insects including but not limited to species belonging to thefamily Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorusguildini, Euschistus servus, Acrosternum hilare, Euschistus heros,Euschistus tristigmus, Acrosternum hilare, Dichelops furcatus, Dichelopsmelacanthus, and Bagrada hilaris (Bagrada Bug)), the family Plataspidae(Megacopta cribraria—Bean plataspid) and the family Cydnidae(Scaptocoris castanea—Root stink bug) and Lepidoptera species includingbut not limited to: diamond-back moth, e.g., Helicoverpa zea Boddie;soybean looper, e.g., Pseudoplusia includens Walker and velvet beancaterpillar e.g., Anticarsia gemmatalis Hübner.

Methods for measuring pesticidal activity are well known in the art.See, for example, Czapla and Lang, (1990) J. Econ. Entomol.83:2480-2485; Andrews, et al., (1988) Biochem. J. 252:199-206; Marrone,et al., (1985) J. of Economic Entomology 78:290-293 and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety. Generally, the protein is mixed and used in feeding assays.See, for example Marrone, et al., (1985) J. of Economic Entomology78:290-293. Such assays can include contacting plants with one or morepests and determining the plant's ability to survive and/or cause thedeath of the pests.

Nematodes include parasitic nematodes such as root-knot, cyst and lesionnematodes, including Heterodera spp., Meloidogyne spp. and Globoderaspp.; particularly members of the cyst nematodes, including, but notlimited to, Heterodera glycines (soybean cyst nematode); Heteroderaschachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode)and Globodera rostochiensis and Globodera pailida (potato cystnematodes). Lesion nematodes include Pratylenchus spp.

Seed Treatment

To protect and to enhance yield production and trait technologies, seedtreatment options can provide additional crop plan flexibility and costeffective control against insects, weeds and diseases. Seed material canbe treated, typically surface treated, with a composition comprisingcombinations of chemical or biological herbicides, herbicide safeners,insecticides, fungicides, germination inhibitors and enhancers,nutrients, plant growth regulators and activators, bactericides,nematocides, avicides and/or molluscicides. These compounds aretypically formulated together with further carriers, surfactants orapplication-promoting adjuvants customarily employed in the art offormulation. The coatings may be applied by impregnating propagationmaterial with a liquid formulation or by coating with a combined wet ordry formulation. Examples of the various types of compounds that may beused as seed treatments are provided in The Pesticide Manual: A WorldCompendium, C. D. S. Tomlin Ed., Published by the British CropProduction Council, which is hereby incorporated by reference.

Some seed treatments that may be used on crop seed include, but are notlimited to, one or more of abscisic acid, acibenzolar-S-methyl,avermectin, amitrol, azaconazole, azospirillum, azadirachtin,azoxystrobin, Bacillus spp. (including one or more of cereus, firmus,megaterium, pumilis, sphaericus, subtilis and/or thuringiensis species),Bradyrhizobium spp. (including one or more of betae, canariense,elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and/oryuanmingense), captan, carboxin, chitosan, clothianidin, copper,cyazypyr, difenoconazole, etidiazole, fipronil, fludioxonil,fluoxastrobin, fluquinconazole, flurazole, fluxofenim, harpin protein,imazalil, imidacloprid, ipconazole, isoflavenoids,lipo-chitooligosaccharide, mancozeb, manganese, maneb, mefenoxam,metalaxyl, metconazole, myclobutanil, PCNB, penflufen, penicillium,penthiopyrad, permethrine, picoxystrobin, prothioconazole,pyraclostrobin, rynaxypyr, S-metolachlor, saponin, sedaxane, TCMTB,tebuconazole, thiabendazole, thiamethoxam, thiocarb, thiram,tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin,triticonazole and/or zinc. PCNB seed coat refers to EPA RegistrationNumber 00293500419, containing quintozen and terrazole. TCMTB refers to2-(thiocyanomethylthio) benzothiazole.

Seed varieties and seeds with specific transgenic traits may be testedto determine which seed treatment options and application rates maycomplement such varieties and transgenic traits in order to enhanceyield. For example, a variety with good yield potential but head smutsusceptibility may benefit from the use of a seed treatment thatprovides protection against head smut, a variety with good yieldpotential but cyst nematode susceptibility may benefit from the use of aseed treatment that provides protection against cyst nematode, and soon. Likewise, a variety encompassing a transgenic trait conferringinsect resistance may benefit from the second mode of action conferredby the seed treatment, a variety encompassing a transgenic traitconferring herbicide resistance may benefit from a seed treatment with asafener that enhances the plants resistance to that herbicide, etc.Further, the good root establishment and early emergence that resultsfrom the proper use of a seed treatment may result in more efficientnitrogen use, a better ability to withstand drought and an overallincrease in yield potential of a variety or varieties containing acertain trait when combined with a seed treatment.

Methods for Killing an Insect Pest and Controlling an Insect Population

In some embodiments methods are provided for killing an insect pest,comprising contacting the insect pest with an insecticidally-effectiveamount of an insecticidal polypeptide of the disclosure. In someembodiments methods are provided for killing an insect pest, comprisingcontacting the insect pest with an insecticidally-effective amount of aPIP-45-1 polypeptide of the embodiments and a PIP-45-2 polypeptide ofthe embodiments, a PIP-64-1 polypeptide of the embodiments and aPIP-64-2 polypeptide of the embodiments, a PIP-74-1 polypeptide of theembodiments and a PIP-74-2 polypeptide of the embodiments, a PIP-75polypeptide of the embodiments and/or a PIP-77 polypeptide of theembodiments.

In some embodiments methods are provided for controlling an insect pestpopulation, comprising contacting the insect pest population with aninsecticidally-effective amount of a recombinant insecticidalpolypeptide of the embodiments. In some embodiments methods are providedfor controlling an insect pest population, comprising contacting theinsect pest population with an insecticidally-effective amount of aPIP-45-1 polypeptide of the embodiments and a PIP-45-2 polypeptide ofthe embodiments, a PIP-64-1 polypeptide of the embodiments and aPIP-64-2 polypeptide of the embodiments, a PIP-74-1 polypeptide of theembodiments and a PIP-74-2 polypeptide of the embodiments, a PIP-75polypeptide of the embodiments and/or a PIP-77 polypeptide of theembodiments. As used herein, “controlling a pest population” or“controls a pest” refers to any effect on a pest that results inlimiting the damage that the pest causes. Controlling a pest includes,but is not limited to, killing the pest, inhibiting development of thepest, altering fertility or growth of the pest in such a manner that thepest provides less damage to the plant, decreasing the number ofoffspring produced, producing less fit pests, producing pests moresusceptible to predator attack or deterring the pests from eating theplant.

In some embodiments methods are provided for controlling an insect pestpopulation resistant to a pesticidal protein, comprising contacting theinsect pest population with an insecticidally-effective amount of arecombinant insecticidal polypeptide of the disclosure. In someembodiments methods are provided for controlling an insect pestpopulation resistant to a pesticidal protein, comprising contacting theinsect pest population with an insecticidally-effective amount of aPIP-45-1 polypeptide of the embodiments and a PIP-45-2 polypeptide ofthe embodiments, a PIP-64-1 polypeptide of the embodiments and aPIP-64-2 polypeptide of the embodiments, a PIP-74-1 polypeptide of theembodiments and a PIP-74-2 polypeptide of the embodiments, a PIP-75polypeptide of the embodiments and/or a PIP-77 polypeptide of theembodiments.

In some embodiments methods are provided for protecting a plant from aninsect pest, comprising expressing in the plant or cell thereof arecombinant polynucleotide encoding an insecticidal polypeptide of thedisclosure. In some embodiments methods are provided for protecting aplant from an insect pest, comprising expressing in the plant or cellthereof a recombinant polynucleotide encoding pesticidal protein of aPIP-45-1 polypeptide of the embodiments and a PIP-45-2 polypeptide ofthe embodiments, a PIP-64-1 polypeptide of the embodiments and aPIP-64-2 polypeptide of the embodiments, a PIP-74-1 polypeptide of theembodiments and a PIP-74-2 polypeptide of the embodiments, a PIP-75polypeptide of the embodiments and/or a PIP-77 polypeptide of theembodiments.

Insect Resistance Management (IRM) Strategies

Expression of B. thuringiensis δ-endotoxins in transgenic corn plantshas proven to be an effective means of controlling agriculturallyimportant insect pests (Perlak, et al., 1990; 1993). However, insectshave evolved that are resistant to B. thuringiensis δ-endotoxinsexpressed in transgenic plants. Such resistance, should it becomewidespread, would clearly limit the commercial value of germplasmcontaining genes encoding such B. thuringiensis δ-endotoxins.

One way to increasing the effectiveness of the transgenic insecticidesagainst target pests and contemporaneously reducing the development ofinsecticide-resistant pests is to use provide non-transgenic (i.e.,non-insecticidal protein) refuges (a section of non-insecticidalcrops/corn) for use with transgenic crops producing a singleinsecticidal protein active against target pests. The United StatesEnvironmental Protection Agency(epa.gov/oppbppdl/biopesticides/pips/bt_corn_refuge_2006.htm, which canbe accessed using the www prefix) publishes the requirements for usewith transgenic crops producing a single Bt protein active againsttarget pests. In addition, the National Corn Growers Association, ontheir website:(ncga.com/insect-resistance-management-fact-sheet-bt-corn, which can beaccessed using the www prefix) also provides similar guidance regardingrefuge requirements. Due to losses to insects within the refuge area,larger refuges may reduce overall yield.

Another way of increasing the effectiveness of the transgenicinsecticides against target pests and contemporaneously reducing thedevelopment of insecticide-resistant pests would be to have a repositoryof insecticidal genes that are effective against groups of insect pestsand which manifest their effects through different modes of action.

Expression in a plant of two or more insecticidal compositions toxic tothe same insect species, each insecticide being expressed at efficaciouslevels would be another way to achieve control of the development ofresistance. This is based on the principle that evolution of resistanceagainst two separate modes of action is far more unlikely than only one.Roush, for example, outlines two-toxin strategies, also called“pyramiding” or “stacking,” for management of insecticidal transgeniccrops. (The Royal Society. Phil. Trans. R. Soc. Lond. B. (1998)353:1777-1786). Stacking or pyramiding of two different proteins eacheffective against the target pests and with little or nocross-resistance can allow for use of a smaller refuge. The USEnvironmental Protection Agency requires significantly less (generally5%) structured refuge of non-Bt corn be planted than for single traitproducts (generally 20%). There are various ways of providing the IRMeffects of a refuge, including various geometric planting patterns inthe fields and in-bag seed mixtures, as discussed further by Roush.

In some embodiments the insecticidal polypeptides of the disclosure areuseful as an insect resistance management strategy in combination (i.e.,pyramided) with other pesticidal proteins include but are not limited toBt toxins, Xenorhabdus sp. or Photorhabdus sp. insecticidal proteins,and the like.

Provided are methods of controlling Lepidoptera and/or Coleoptera insectinfestation(s) in a transgenic plant that promote insect resistancemanagement, comprising expressing in the plant at least two differentinsecticidal proteins having different modes of action.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management the at least one of the insecticidal proteinscomprise an insecticidal polypeptide of the disclosure insecticidal toinsects in the order Lepidoptera and/or Coleoptera.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management comprise expressing in the transgenic plant aninsecticidal polypeptide of the disclosure and a Cry proteininsecticidal to insects in the order Lepidoptera and/or Coleopterahaving different modes of action.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management comprise in the transgenic plant a PIP-45-1polypeptide of the embodiments and a PIP-45-2 polypeptide of theembodiments, a PIP-64-1 polypeptide of the embodiments and a PIP-64-2polypeptide of the embodiments, a PIP-74-1 polypeptide of theembodiments and a PIP-74-2 polypeptide of the embodiments, a PIP-75polypeptide of the embodiments or a PIP-77 polypeptide of theembodiments and a Cry protein insecticidal to insects in the orderLepidoptera and/or Coleoptera having different modes of action.

Also provided are methods of reducing likelihood of emergence ofLepidoptera and/or Coleoptera insect resistance to transgenic plantsexpressing in the plants insecticidal proteins to control the insectspecies, comprising expression of an insecticidal polypeptide of thedisclosure insecticidal to the insect species in combination with asecond insecticidal protein to the insect species having different modesof action.

Also provided are methods of reducing likelihood of emergence ofLepidoptera and/or Coleoptera insect resistance to transgenic plantsexpressing in the plants insecticidal proteins to control the insectspecies, comprising expression of a PIP-45-1 polypeptide of theembodiments and a PIP-45-2 polypeptide of the embodiments, a PIP-64-1polypeptide of the embodiments and a PIP-64-2 polypeptide of theembodiments, a PIP-74-1 polypeptide of the embodiments and a PIP-74-2polypeptide of the embodiments, a PIP-75 polypeptide of the embodimentsor a PIP-77 polypeptide of the embodiments, insecticidal to the insectspecies in combination with a second insecticidal protein to the insectspecies having different modes of action.

Also provided are means for effective Lepidoptera and/or Coleopterainsect resistance management of transgenic plants, comprisingco-expressing at high levels in the plants two or more insecticidalproteins toxic to Lepidoptera and/or Coleoptera insects but eachexhibiting a different mode of effectuating its killing activity,wherein the two or more insecticidal proteins comprise an insecticidalpolypeptide of the disclosure and a Cry protein. Also provided are meansfor effective Lepidoptera and/or Coleoptera insect resistance managementof transgenic plants, comprising co-expressing at high levels in theplants two or more insecticidal proteins toxic to Lepidoptera and/orColeoptera insects but each exhibiting a different mode of effectuatingits killing activity, wherein the two or more insecticidal proteinscomprise a PIP-45-1 polypeptide of the embodiments and a PIP-45-2polypeptide of the embodiments, a PIP-64-1 polypeptide of theembodiments and a PIP-64-2 polypeptide of the embodiments, a PIP-74-1polypeptide of the embodiments and a PIP-74-2 polypeptide of theembodiments, a PIP-75 polypeptide of the embodiments or a PIP-77polypeptide of the embodiments, and a Cry protein.

In addition, methods are provided for obtaining regulatory approval forplanting or commercialization of plants expressing proteins insecticidalto insects in the order Lepidoptera and/or Coleoptera, comprising thestep of referring to, submitting or relying on insect assay binding datashowing that the insecticidal polypeptide of the disclosure does notcompete with binding sites for Cry proteins in such insects. Inaddition, methods are provided for obtaining regulatory approval forplanting or commercialization of plants expressing proteins insecticidalto insects in the order Lepidoptera and/or Coleoptera, comprising thestep of referring to, submitting or relying on insect assay binding datashowing that the PIP-45-1 polypeptide of the embodiments & the PIP-45-2polypeptide of the embodiments, the PIP-64-1 polypeptide of theembodiments & the PIP-64-2 polypeptide of the embodiments, the PIP-74-1polypeptide of the embodiments & the PIP-74-2 polypeptide of theembodiments, the PIP-75 polypeptide of the embodiments or the PIP-77polypeptide of the embodiments does not compete with binding sites forCry proteins in such insects.

Methods for Increasing Plant Yield

Methods for increasing plant yield are provided. The methods compriseproviding a plant or plant cell expressing a polynucleotide encoding thepesticidal polypeptide sequence disclosed herein and growing the plantor a seed thereof in a field infested with a pest against which thepolypeptide has pesticidal activity. In some embodiments, thepolypeptide has pesticidal activity against a Lepidopteran, Coleopteran,Dipteran, Hemipteran or nematode pest, and the field is infested with aLepidopteran, Hemipteran, Coleopteran, Dipteran or nematode pest.

As defined herein, the “yield” of the plant refers to the quality and/orquantity of biomass produced by the plant. “Biomass” as used hereinrefers to any measured plant product. An increase in biomass productionis any improvement in the yield of the measured plant product.Increasing plant yield has several commercial applications. For example,increasing plant leaf biomass may increase the yield of leafy vegetablesfor human or animal consumption. Additionally, increasing leaf biomasscan be used to increase production of plant-derived pharmaceutical orindustrial products. An increase in yield can comprise any statisticallysignificant increase including, but not limited to, at least a 1%increase, at least a 3% increase, at least a 5% increase, at least a 10%increase, at least a 20% increase, at least a 30%, at least a 50%, atleast a 70%, at least a 100% or a greater increase in yield compared toa plant not expressing the pesticidal sequence.

In specific methods, plant yield is increased as a result of improvedpest resistance of a plant expressing an insecticidal polypeptide of thedisclosure disclosed herein. Expression of the insecticidal polypeptideof the disclosure results in a reduced ability of a pest to infest orfeed on the plant, thus improving plant yield.

Methods of Processing

Further provided are methods of processing a plant, plant part or seedto obtain a food or feed product from a plant, plant part or seedcomprising an insecticidal polypeptide of the disclosure. The plants,plant parts or seeds provided herein, can be processed to yield oil,protein products and/or by-products that are derivatives obtained byprocessing that have commercial value. Non-limiting examples includetransgenic seeds comprising a nucleic acid molecule encoding aninsecticidal polypeptide of the disclosure which can be processed toyield soy oil, soy products and/or soy by-products.

“Processing” refers to any physical and chemical methods used to obtainany soy product and includes, but is not limited to, heat conditioning,flaking and grinding, extrusion, solvent extraction or aqueous soakingand extraction of whole or partial seeds

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTALS Example 1 Insect Feeding Assays

Insecticidal activity bioassay screens were conducted on the clearedlysate to evaluate the effects of the insecticidal proteins on a varietyof Lepidoptera species (European corn borer (Ostrinia nubilalis), cornearworm (Helicoverpa zea), black cutworm (Agrotis ipsilon), fallarmyworm (Spodoptera frugiperda), Soybean looper (Pseudoplusiaincludens) and Velvet bean caterpillar (Anticarsia gemmatalis)), aColeoptera specie (Western corn rootworm (Diabrotica virgifera), and twoHemiptera species, Lygus hesperus and Nezara viridula (Southern GreenStinkbug).

Lepidoptera Assays

Lepidoptera feeding assays were conducted on an artificial dietcontaining the cleared lysates of bacterial strains in a 96 well plateset up. The cleared lysate was incorporated with theLepidopteran-specific artificial diet in a ratio of 20 ul cleared lysateand 40 ul of diet mixture. Two to five neonate larvas were placed ineach well to feed for 5 days. Results were expressed as positive forlarvae reactions such as stunting and/or mortality. Results wereexpressed as negative if the larvae were similar to the negative controlthat is feeding diet to which the above buffer only has been applied.Each cleared lysate was assayed on European corn borer (Ostrinianubilalis), corn earworm (Helicoverpa zea), black cutworm (Agrotisipsilon), fall armyworm (Spodoptera frugiperda), Soybean looper(Pseudoplusia includens) and Velvet bean caterpillar (Anticarsiagemmatalis). A series of concentrations of the purified protein samplewas assayed against those insects and concentrations for 50% mortality(LC50) or inhibition of 50% of the individuals (IC50) were calculated.

Coleoptera Assays

Coleoptera feeding assays were conducted on an artificial dietcontaining the cleared lysates of bacterial strains in a 96 well plateset up. The cleared lysate was incorporated with thecoleopteran-specific artificial diet in a ratio of 10 ul cleared lysateand 50 ul of diet mixture. Two to five Western corn rootworm (Diabroticavirgifera) neonate larva were placed in each well to feed for 5 days.Results were expressed as positive for larvae reactions such as stuntingand/or mortality. Results were expressed as negative if the larvae weresimilar to the negative control that is feeding diet to which the abovebuffer only has been applied. A series of concentrations of the purifiedprotein sample was assayed against those insects and concentrations for50% mortality (LC50) or inhibition of 50% of the individuals (IC50) werecalculated.

Lygus (Lygus Hesperus) Bioassay

20 ul of the cleared lysate samples were mixed with 75 ul Lygus diet(Bio-Serv F9644B) in each well of a 96 well bioassay plate (BD Falcon353910) and covered with a sheet of Parafilm. A variable numbers ofLygus hesperus second instar nymphs (2 to 7) were placed into each wellof a 96 well filter plate. The sample plate was then flipped on to thefilter plate and held together with rubber bands. The assay was run fourdays at 25° C. and then was scored for insect mortality and/or stuntingof insect growth. A series of concentrations of the purified proteinsample was assayed against those insects and concentrations for 50%mortality (LC50) or inhibition of 50% of the individuals (IC50) werecalculated.

Southern Green Stinkbug (Nezara Viridula) and Brown Marmorated Stinkbug(Halyomorpha Haly) Bioassay

40 ul of the cleared lysate samples were mixed with 360 ul of Lygus diet(Bio-Serv F9644B) in Parafilm® packets. 10 to 15 newly molted instarnymphs were placed in polystyrene Petri dishes (100 mm×20 mm) lined withmoist Whatman® filter paper (100 mm diameter). Included in the dish wasa water source. The bioassay was incubated at 25° C. in the dark forfour days. The bioassay was scored for mortality and stunting. Togenerate ILC50 or LC50 data, a series of concentrations of purifiedproteins were assayed against insects and the concentration at which 50%of the insects experienced severe damage was the ILC50 and theconcentration at which 50% of insects were dead was the LC50.

Colorado Potato Beetle (Leptinotarsa Decemlineata) Bioassay

20 ul of cleared lysate samples were mixed with 75 ul of modifiedColeopteran diet (Bio-Serv F9800B) in each well of a 96 well bioassayplate (BD Falcon 353910) and allowed to solidify. A single neonate larvawas placed in each well and the plate sealed with a Mylar® covering.Holes were punched in the Mylar® sheet and the plate incubated at 25° C.with no light for four days. The bioassay was scored for mortalityand/or stunting.

Example 2 Identification of Insecticidal Active Strains

Insecticidal activities against SBL, CEW, BCW, VBC, ECB, Lygus, SGSB,and WCRW were observed from a clear cell lysate of bacterial strainsgrown in either LB medium (10 g/L tryptone, 5 g/L yeast extract, and 10g/L NaCl) or TSB (Tryptic Soy Broth) medium (17 g/L tryptone, 3 g/Lsoytone, 2.5 g/L dextrose, 2.5 g/L K₂HPO₄ and 5 g/L NaCl) and culturedovernight at 26° C. with shaking at 250 rpm. This insecticidal activityexhibited heat and proteinase sensitivity indicating proteinaceousnature. Active strains and their insecticidal activities were listed inTable 5.

TABLE 5 Strain Species Target insects Gene Seq. No. LBV5480 PseudomonasWCRW PIP-45Aa-1/2 SEQ ID NO: 1/ brenneri SEQ ID NO: 2 LBV9691Pseudomonas WCRW PIP-64Aa-1 SEQ ID NO: 53 brenneri alone LBV9691Pseudomonas SBL, CEW, BCW, VBC, PIP-64Aa-1/2 SEQ ID NO: 53/ brenneriECB, Lygus, SGSB SEQ ID NO: 54 SS135B4b Pseudomonas WCRW PIP-74Aa-1/2SEQ ID NO: 73/ rhodesiae SEQ ID NO: 74 LBV6019 Pseudomonas WCRW PIP-75AaSEQ ID NO: 79 antarctica SSP344E5a Pseudomonas WCRW PIP-77Aa SEQ ID NO:88 chlororaphis

Example 3 Species Identification and Genome Sequencing of Active Strains

Genomic DNA from active strains was extracted with a Sigma® BacterialGenomic DNA Extraction Kit (Cat #NA2110-KT, Sigma-Aldrich, PO Box 14508,St. Louis, Mo. 63178) according to the manufactures' instructions. TheDNA concentration was determined using a NanoDrop™ Spectrophotometer(Thermo Scientific, 3411 Silverside Road, Bancroft Building, Suite 100,Wilmington, Del. 19810) and the genomic DNA was diluted to 40 ng/ul withsterile water. A 25 ul PCR reaction was set up by combining 80 nggenomic DNA, 2 ul (5 uM) 16S ribosomal DNA primers TACCTTGTTACGACTT (SEQID NO: 216) and AGAGTTTGATCMTGGCTCAG (SEQ ID NO: 217), 1 ul 10 cmM dNTP,1× Phusion® HF buffer, and 1 unit of Phusion® High-Fidelity DNAPolymerase (New England Biolabs, Cat #M0530L, 240 County Road, Ipswich,Mass. 01938-2723). The PCR reaction was run in MJ Research PTC-200Thermo Cycler (Bio-Rad Laboratories, Inc., 1000 Alfred Nobel Drive,Hercules, Calif., 94547, USA) with the following program: 96° C. 1 min;30 cycles of 96° C. 15 seconds, 52° C. 2 minutes and 72° C. 2 minutes;72° C. 10 minutes; and hold on 4° C. The PCR products were purified withQiaquick® DNA purification Kit (Cat #28104, QIAGEN Inc., 27220 TurnberryLane, Valencia, Calif. 91355). The purified PCR sample was DNA sequencedand the resulting 16S ribosomal DNA sequence was BLAST searched againstthe NCBI database. The top hits indicated the species of the strain (seeTable 5).

Genomic DNA of active strains was also prepared according to a libraryconstruction protocol developed by Illumina and sequenced using theIllumina MiSeq™. The nucleic acid contig sequences were assembled andopen reading frames were generated.

Example 4 Identification of Insecticidal Proteins by LC-MS/MS

All insecticidal proteins were fractionated and enriched as described.For identification candidate protein bands were excised, digested withtrypsin and analyzed by nano-liquid chromatography/electrospray tandemmass spectrometry (nano-LC/ESI-MS/MS) on a Thermo Q Exactive™ Orbitrap™mass spectrometer (Thermo Fisher Scientific) interfaced with anEksigent® NanoLC-1D™ Plus nanoLC™ system (AB Sciex). Ten product ionspectra were collected in an information dependent acquisition modeafter a MS1 survey scan.

Protein identification was done by database searches using Mascot(Matrix Science). The searches were done against the in-house databaseBacteria-Plus, which combines all bacterial protein sequences andkeratin sequences derived from the NCBI non-redundant database (nr) aswell as in-house protein sequences.

Example 5 Isolation and Identification of Insecticidal ProteinsIsolation and Identification of PIP-45-Aa-1 and PIP-45-Aa-2

Insecticidal activity against WCRW (Diabrotica virgifera) was observedfrom a clear cell lysate of from Pseudomonas brenneri strain LBV 5480grown in Nutrient Broth (Peptone—5 g/L, Meat extract—1 g/L, Yeastextract—2 g/L, Sodium chloride—5 g/L) and cultured overnight at 26° C.with shaking at 250 rpm. This insecticidal activity exhibited heat andprotease sensitivity indicating proteinaceous nature.

Cell pellets of LBV 5480 were homogenized at 20,000 psi afterre-suspension in Tris buffer, pH 8. The crude lysate was cleared bycentrifugation and loaded onto a HiTrap® Q-FF column (GE Healthcare).Bound protein was eluted with a linear sodium chloride gradient andfractionated. Fractions containing protein of interest were pooled andadjusted to 1 M ammonium sulfate concentration in 50 mM Tris (buffer A).This material was loaded onto a Phenyl Sepharose HP HiTrap® column (GEHealthcare) equilibrated in buffer A. Active protein was eluted with alinear gradient from 1 M to 0 M ammonium sulfate and further purified bysize exclusion chromatography. For this the Phenyl-pool was concentratedand loaded onto a Superdex® 200 column (GE Healthcare), equilibrated in20 mM Tris, 150 mM NaCl, pH 8. SDS-PAGE analysis of fractions with WCRWactivity showed 2 predominant bands after staining with Coomassie® Bluedye. LC-MS/MS was used to identify two novel genes encoded by strain LBV5480. These genes form an operon and both gene products are required forinsecticidal activity as confirmed with recombinant protein. Theseproteins were designated as PIP-45-Aa-1 (SEQ ID NO: 1) and PIP-45-Aa-2(SEQ ID NO: 2).

Isolation and Identification of PIP-64-Aa-1 and PIP-64-Aa-2

Insecticidal activity against WCRW (Diabrotica virgifera) and soybeanlooper (SBL Chrysodeixis includes) was observed from a clear cell lysateof Pseudomonas brenneri strain LBV 9691 grown in 2× YT medium (16 g/LTryptone, 10 g/L Yeast Extract, 5 g/L NaCl) and cultured for 3 days at26° C. with shaking at 250 rpm. This insecticidal activity exhibitedheat and protease sensitivity indicating proteinaceous nature.

Growth conditions and insect activity varied greatly. Higher activityalso correlated with higher expression levels of a protein band of ˜28kDa, detectable by SDS-PAGE. To further confirm this candidate band,cell pellets of LVB 9691 were homogenized at 30,000 psi afterre-suspension in 20 mM Tris buffer, pH 8. The crude lysate was clearedby centrifugation and loaded onto a Superdex® 75 column (GE Healthcare).WCRW and SBL activities were strongly associated with the 28 kDa band inthe elution fractions. The protein band was identified LC-MS/MS.Database search identified two novel proteins of similar size encoded inan operon by strain LBV 9691, designated PIP-64-Aa-1 (SEQ ID NO: 53) andPIP-64-Aa-2 (SEQ ID NO: 54). Recombinant expression showed that at theconcentrations tested both proteins are required for activity againstLepidopteran and Hemipteran species. Activity was found to be optimal ata molar ratio of 5:1 of PIP-64Aa-1 (SEQ ID NO: 53) and PIP-64Aa-2 (SEQID NO: 54), respectively. PIP-64-Aa-1 (SEQ ID NO: 53) alone wassufficient for WCRW activity.

Isolation and Identification of PIP-74-Aa-1 and PIP-74-Aa-2

Insecticidal activity against WCRW (Diabrotica virgifera) was observedfrom a clear cell lysate of Pseudomonas brenneri strain SS135B4 grown in2× YT medium and cultured for 2 days at 26° C. with shaking at 250 rpm.This insecticidal activity exhibited heat and protease sensitivityindicating proteinaceous nature.

Cell pellets of SS135B4 were homogenized at 30,000 psi afterre-suspension in 25 mM Tris buffer, pH 9. The crude lysate was clearedby centrifugation, adjusted to 0.5 M ammonium sulfate and loaded onto aPhenyl Sepharose FF column (GE Healthcare). WCRW active protein waseluted with a linear gradient to 0 M ammonium sulfate, pooled anddialyzed into 50 mM sodium acetate buffer, pH 5. The adjusted pool wasthen loaded onto an S-Sepharose FF column (GE Healthcare) which wasequilibrated with the same buffer. The unbound protein fraction,containing WCRW active protein, was buffer exchanged to 50 mM CAPS, pH10, loaded onto a MonoQ® column (GE Healthcare) and eluted with a linearsodium chloride gradient in 50 mM CAPS, pH10. SDS-PAGE analysis of thesefractions showed several bands after staining with Coomassie® Blue dye.The protein bands were excised and identified by LC-MS/MS. Databasesearch identified two novel proteins encoded in an operon by strainSS135B4, designated PIP-74-Aa-1 (SEQ ID NO: 73) and PIP-74-Aa-2 (SEQ IDNO: 74), respectively. Recombinant expression showed that at theconcentrations tested both proteins are required for activity againstWCRW.

Isolation and Identification of PIP-75-Aa

Insecticidal activity against WCRW (Diabrotica virgifera) was observedfrom a clear cell lysate of Pseudomonas antarctica LBV 6019 grown in 2×YT medium for 1 day at 26° C. with shaking at 250 rpm. This insecticidalactivity exhibited heat and protease sensitivity indicatingproteinaceous nature.

Cell pellets of LBV 6019 were homogenized at 30,000 psi afterre-suspension in 25 mM Tris buffer, pH 8.5. The crude lysate was clearedby centrifugation and loaded onto a POROS® Q column (Life Technologies).The unbound protein fraction, containing WCRW active protein, was bufferexchanged by dialysis against 10 mM MES, pH 6 and then loaded onto aHiTrap® S-HP column (GE Healthcare) and eluted with a linear sodiumchloride gradient. Fractions containing active protein were pooled,buffer adjusted and subjected to a repeated anion exchange step at pH8.5. The unbound fraction was buffer exchanged again before a finalfractionation step on a Mono Se column (GE Healthcare), equilibratedwith 20 mM MES, pH 6. Several active fractions were obtained afterelution with a linear gradient to 0.3 M NaCl. SDS-PAGE analysis offractions with WCRW activity showed several predominant bands afterstaining with Coomassie®Blue dye. The protein bands were excised andidentified through LC-MS/MS.

Database search revealed 3 novel gene candidates encoded by strain LBV6019. Cloning and recombinant expression confirmed the insecticidalactivity of one of the candidates. This protein was designated asPIP-75-Aa (SEQ ID NO: 79).

Isolation and Identification of PIP-77-Aa

Insecticidal activity against WCRW (Diabrotica virgifera) was observedfrom a clear cell lysate of Pseudomonas chlororaphis strain SS344E5grown in Tryptic Soy broth (TSB, peptone from casein 15 g/L; peptonefrom soymeal 5 g/L; sodium chloride 5.0 g/L) for 1 day at 26° C. withshaking at 250 rpm. This insecticidal activity exhibited heat andprotease sensitivity indicating proteinaceous nature.

Cell pellets of SS344E5 were homogenized at 30,000 psi afterre-suspension in 25 mM Tris buffer, pH 8.5. The crude lysate was clearedby centrifugation and loaded onto a POROS® Q column (Life Technologies).The unbound protein fraction, containing WCRW active protein, wasdialyzed against 10 mM MES, pH 6, loaded onto a HiTrap® S-HP column (GEHealthcare), and eluted with a linear sodium chloride gradient to 0.5 M.Fractions containing WCRW active protein were pooled and furtherseparated by size exclusion chromatography using a Superdex® 75 column(GE Healthcare). SDS-PAGE analysis of fractions with WCRW activityshowed a predominant band of 7 kDa after staining with Coomassie® Bluedye. LC-MS/MS was used to identify two novel genes encoded by strainSS344E5. Cloning and recombinant expression confirmed the insecticidalactivity of this gene product, which was designated as PIP-77-Aa (SEQ IDNO: 88).

Example 6 Identification of Homologs

Genomic DNA was extracted from various internal strains, the species wasidentified and the genome was sequences as described in Example 3. Geneidentities may be determined by conducting BLAST (Basic Local Alignment20 Search Tool; Altschul, et al., (1993) J. Mol. Biol. 215:403-410; seealso ncbi.nlm.nih.gov/BLAST/, which can be accessed using the wwwprefix) searches under default parameters for similarity to sequencescontained in the internal genomes and in the publically available BLAST“nr” database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the 25 SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). The polypeptide sequences of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 73, SEQ IDNO: 74, SEQ ID NO: 79 and SEQ ID NO: 88 were analyzed.

Table 6 shows the PIP-45-1 polypeptide and PIP-45-2 polypeptide homologsidentified, sequence identification numbers for each and the bacterialstrains they were identified from. Table 7 shows the percent sequenceidentity between the PIP-45-1 polypeptide homologs. FIG. 1a-1m shows anamino acid sequence alignment of the PIP-45-1 polypeptide homologs.

TABLE 6 Gene Sequence # Source Species Activity PIP-45Aa-1 SEQ ID NO: 1LBV5480 Pseudomonas yes PIP-45Aa-2 SEQ ID NO: 2 brenneri PIP-45Ab-1 SEQID NO: 3 LBV2335-5 (1-2aa difference); Pseudomonas sp. n.d. ^(†)PIP-45Ab-2 SEQ ID NO: 4 LBV8526-5; NCBI hypothetical protein Ag1ZP_10476580) and ZP_10476581 PIP-45Ac-1 SEQ ID NO: 5 NCBI hypotheticalprotein Pseudomonas sp. n.d. PIP-45Ac-2 SEQ ID NO: 6 ZP_10430003 andZP_10430004 PAMC 25886 PIP-45Ad-1 SEQ ID NO: 7 NCBI hypothetical proteinPseudomonas sp. yes PIP-45Ad-2 SEQ ID NO: 8 ZP_10430003,JGI_XylAfBL_518010 PAMC 25886 PIP-45Ae-1 SEQ ID NO: 9 internal strainSSP143E2; LBV9925-5; Pseudomonas n.d. EMBL K1AVN2_PSEFL; NCBIfluorescens ZP_1559991; PIP-45Ae-2 SEQ ID NO: 10 internal strainSSP143E2; EMBL K1B453_PSEFL; NCBI ZP_15599912; PIP-45Af-1 SEQ ID NO: 11NCBI hypothetical protein Pseudomonas sp. n.d. WP_017475319 PAMC 26793PIP-45Af-2 SEQ ID NO: 12 NCBI hypothetical protein WP_017475320PIP-45Ba-1 SEQ ID NO: 13 NCBI hypothetical protein PPs_2675 Pseudomonasyes PIP-45Ba-2 SEQ ID NO: 14 (YP_004702108.1) and PPS_2674 putida(YP_004702107.1) PIP-45Bb-1 SEQ ID NO: 15 JGI - AECFG_342250hypothetical Fungus garden n.d. protein combined PIP-45Bb-2 SEQ ID NO:16 JGI - AECFG_342240 hypothetical protein PIP-45Bc-1 SEQ ID NO: 17internal collectionSSP145B2; Pseudomonas n.d. PIP-45Bc-2 SEQ ID NO: 18SSP469C8a monteilii PIP-45Bd-1 SEQ ID NO: 19 internal collection -SS160F12; Pseudomonas yes PIP-45Bd-2 SEQ ID NO: 20 SSP165H7; SS153D5a;SS165D11-2; monteilii JH23144-1; PIP-45Be-1 SEQ ID NO: 21 LBV9691Pseudomonas yes PIP-45Be-2 SEQ ID NO: 22 brenneri PIP-45Bf-1 SEQ ID NO:23 LBV11272; LBV11224: LBV10925 Pseudomonas n.d. PIP-45Bf-2 SEQ ID NO:24 gessardii PIP-45Bg-1 SEQ ID NO: 25 NCBI B479_12925 Pseudomonas n.d.PIP-45Bg-2 SEQ ID NO: 26 NCBI B479_12920 putida PIP-45Bh-1 SEQ ID NO: 27internal collection - SSP339E12-1 Pseudomonas n.d. PIP-45Bh-2 SEQ ID NO:28 plecoglossicida PIP-45Bi-1 SEQ ID NO: 29 internal collection -SSP340D9a Pseudomonas n.d. PIP-45Bi-2 SEQ ID NO: 30 putida PIP-45Bj-1SEQ ID NO: 31 internal collection - JH27606-2, Pseudomonas n.d.PIP-45Bj-2 SEQ ID NO: 32 SSP4C8 putida PIP-45Bk-1 SEQ ID NO: 33 internalcollection - SSP4E8 Pseudomonas n.d. PIP-45Bk-2 SEQ ID NO: 34 monteiliiPIP-45Bl-1 SEQ ID NO: 232 internal collection - JH59565-1; NCBIPseudomonas sp. n.d. PIP-45Bl-2 SEQ ID NO: 233 YP_008763564 andWP_023380724 VLB120 hypothetical proteins PIP-45Bm-1 SEQ ID NO: 234internal collection - JH58750-1 Pseudomonas n.d. PIP-45Bm-2 SEQ ID NO:235 putida PIP-45Ca-1 SEQ ID NO: 35 internal collection - SSi43B5;Pseudomonas poae yes PIP-45Ca-2 SEQ ID NO: 36 SSi44A10; SSP259D11-1;SSP429D11a; SSP429D6a; SS143D2; LBV8661 (2aa difference for 45-2)PIP-45Cb-1 SEQ ID NO: 37 JGI - hypothetical protein Mountain Pine yesPIP-45Cb-2 SEQ ID NO: 38 DPOB_377060 and DPOB_377050 Beetle microbialcommunities PIP-45Cc-1 SEQ ID NO: 39 internal collection - SS137B2Pseudomonas n.d. PIP-45Cc-2 SEQ ID NO: 40 trivialis PIP-45Cd-1 SEQ IDNO: 41 NCBI-ZP_11188561 Pseudomonas sp. n.d. PIP-45Cd-2 SEQ ID NO: 42NCBI-ZP_11188562 R81 PIP-45Ce-1 SEQ ID NO: 43 internal collection -SSP493B7b Pseudomonas n.d. PIP-45Ce-2 SEQ ID NO: 44 libanensisPIP-45Cf-1 SEQ ID NO: 236 internal collection - SSP557A12-2 Pseudomonaspoae n.d. PIP-45Cf-2 SEQ ID NO: 237 Pseudomonas poae n.d. PIP-45Da-1 SEQID NO: 45 internal active strain - SSP347B8a Pseudomonas n.d. PIP-45Da-2SEQ ID NO: 46 asplenii PIP-45Db-1 SEQ ID NO: 47 NCBI-ZP_11115718Thalassospira n.d. PIP-45Db-2 SEQ ID NO: 48 NCBI-ZP_11115719 xiamenensisPIP-45Ea-1 SEQ ID NO: 49 NCBI hypothetical protein Pden_4642 Paracoccusyes PIP-45Ea-2 SEQ ID NO: 50 (YP_918399.1) and Pden_4641 denitrificansPD1222 (YP_918398.1) PIP-45Ga-1 SEQ ID NO: 51 NCBI hypothetical proteinCellvibrio japonicus no PIP-45Ga-2 SEQ ID NO: 52 YP_001984231.1 andUeda107 YP_001984230.1 ^(†) n.d. = not determined

TABLE 7 PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- 45Ab-1 45Ac-145Ad-1 45Ae-1 45Af-1 45Ba-1 45Bb-1 45Bc-1 45Bd-1 SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 3 NO: 5 NO: 7 NO: 9 NO: 11NO: 13 NO: 15 NO: 17 NO: 19 PIP-45Aa-1 98.6 96.5 96.9 98.6 99.0 88.688.4 88.4 87.9 PIP-45Ab-1 — 96.5 97.2 99.7 98.3 88.1 87.9 87.9 87.7PIP-45Ac-1 — — 99.0 96.5 96.0 87.2 87.0 87.0 87.6 PIP-45Ad-1 — — — 97.296.4 87.4 87.2 87.2 87.7 PIP-45Ae-1 — — — — 98.3 88.1 87.9 87.9 87.7PIP-45Af-1 — — — — — 88.6 88.4 88.4 87.9 PIP-45Ba-1 — — — — — — 99.199.3 95.5 PIP-45Bb-1 — — — — — — — 99.1 95.2 PIP-45Bc-1 — — — — — — — —95.2 PIP-45Bd-1 — — — — — — — — — PIP-45Be-1 — — — — — — — — —PIP-45Bf-1 — — — — — — — — — PIP-45Bg-1 — — — — — — — — — PIP-45Bh-1 — —— — — — — — — PIP-45Bi-1 — — — — — — — — — PIP-45Bj-1 — — — — — — — — —PIP-45Bk-1 — — — — — — — — — PIP-45Bl-1 — — — — — — — — — PIP-45Bm-1 — —— — — — — — — PIP-45Ca-1 — — — — — — — — — PIP-45Cb-1 — — — — — — — — —PIP-45Cc-1 — — — — — — — — — PIP-45Cd-1 — — — — — — — — — PIP-45Ce-1 — —— — — — — — — PIP-45Cf-1 — — — — — — — — — PIP-45Da-1 — — — — — — — — —PIP-45Db-1 — — — — — — — — — PIP-45Ea-1 PIP- PIP- PIP- PIP- PIP- PIP-PIP- PIP- PIP- 45Be-1 45Bf-1 45Bg-1 45Bh-1 45Bi-1 45Bj-1 45Bk-1 45Bl-145Bm-1 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDNO: 21 NO: 23 NO: 25 NO: 27 NO: 29 NO: 31 NO: 33 NO: 232 NO: 234PIP-45Aa-1 87.1 87.5 87.7 88.4 88.2 86.0 86.2 86.5 87.4 PIP-45Ab-1 86.687.5 87.2 87.9 87.7 86.2 86.2 86.5 87.4 PIP-45Ac-1 85.9 87.0 86.4 87.086.9 85.3 85.8 86.0 86.7 PIP-45Ad-1 86.1 87.2 86.5 87.2 87.0 85.8 85.386.0 86.7 PIP-45Ae-1 86.8 87.7 87.2 87.9 87.7 85.8 86.0 86.7 87.5PIP-45Af-1 86.6 87.0 87.7 88.4 88.2 86.0 85.8 85.8 87.4 PIP-45Ba-1 84.786.3 98.6 99.0 99.1 92.2 91.0 91.7 93.6 PIP-45Bb-1 84.4 86.0 99.1 98.899.7 91.7 90.8 91.5 93.8 PIP-45Bc-1 84.4 85.8 98.6 98.6 99.1 91.9 91.091.9 93.3 PIP-45Bd-1 84.2 86.3 94.6 95.3 95.2 92.9 90.0 90.8 92.6PIP-45Be-1 — 92.6 84.2 85.1 84.7 81.6 83.8 82.3 83.0 PIP-45Bf-1 — — 85.586.5 86.0 83.6 86.0 84.3 85.3 PIP-45Bg-1 — — — 98.3 99.5 91.2 90.3 91.092.9 PIP-45Bh-1 — — — — 98.8 92.0 91.2 91.2 93.3 PIP-45Bi-1 — — — — —91.7 90.8 91.5 93.4 PIP-45Bj-1 — — — — — — 88.6 87.9 90.3 PIP-45Bk-1 — —— — — — — 90.1 90.8 PIP-45Bl-1 — — — — — — — — 91.0 PIP-45Bm-1 — — — — —— — — — PIP-45Ca-1 — — — — — — — — — PIP-45Cb-1 — — — — — — — — —PIP-45Cc-1 — — — — — — — — — PIP-45Cd-1 — — — — — — — — — PIP-45Ce-1 — —— — — — — — — PIP-45Cf-1 — — — — — — — — — PIP-45Da-1 — — — — — — — — —PIP-45Db-1 — — — — — — — — — PIP-45Ea-1 PIP- PIP- PIP- PIP- PIP- PIP-PIP- PIP- PIP- PIP- 45Ca-1 45Cb-1 45Cc-1 45Cd-1 45Ce-1 45Cf-1 45Da-145Db-1 45Ea-1 45Ga-1 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID NO: 35 NO: 37 NO: 39 NO: 41 NO: 43 NO: 236 NO: 45NO: 47 NO: 49 NO: 51 PIP-45Aa-1 77.9 77.3 76.8 76.8 77.9 78.5 65.1 63.559.8 38.8 PIP-45Ab-1 77.9 76.8 76.5 76.8 77.3 78.2 65.1 63.9 59.8 38.8PIP-45Ac-1 77.2 77.0 76.9 76.7 77.4 77.7 64.3 63.9 59.1 38.0 PIP-45Ad-177.0 76.5 76.3 76.0 76.9 77.7 64.0 63.7 59.0 37.9 PIP-45Ae-1 77.9 77.276.6 76.9 77.7 78.4 65.3 64.0 59.9 39.0 PIP-45Af-1 77.2 76.6 76.1 76.177.2 77.9 65.5 63.5 59.9 38.6 PIP-45Ba-1 77.3 76.8 76.5 76.1 77.0 78.067.0 65.1 59.9 38.4 PIP-45Bb-1 77.3 76.8 76.5 76.1 77.2 78.0 66.8 64.959.8 38.4 PIP-45Bc-1 77.3 76.8 76.5 76.1 77.0 78.0 67.5 65.1 60.1 38.2PIP-45Bd-1 77.0 76.1 75.8 75.4 76.5 77.2 66.5 64.4 59.4 38.3 PIP-45Be-175.6 73.9 73.4 73.9 74.2 75.3 65.4 63.1 57.9 38.8 PIP-45Bf-1 77.4 76.076.0 75.5 76.0 76.9 66.7 64.2 59.6 37.9 PIP-45Bg-1 76.8 76.3 75.8 75.476.5 77.5 66.7 64.4 59.7 38.3 PIP-45Bh-1 77.7 77.2 76.8 76.5 77.5 78.267.0 64.7 60.1 38.5 PIP-45Bi-1 77.3 76.8 76.3 76.0 77.0 78.0 67.0 64.960.1 38.3 PIP-45Bj-1 77.0 75.8 75.4 75.8 75.4 77.2 66.1 64.4 59.6 37.7PIP-45Bk-1 77.7 76.3 76.1 76.0 76.1 76.8 66.1 64.4 59.4 39.4 PIP-45Bl-177.3 76.6 77.0 76.3 76.6 77.9 64.6 64.4 59.6 39.1 PIP-45Bm-1 76.5 76.076.3 75.6 76.5 77.0 66.0 64.8 60.2 38.7 PIP-45Ca-1 — 95.3 92.7 94.3 94.698.1 64.9 66.8 60.8 38.7 PIP-45Cb-1 — — 94.3 96.0 97.4 95.3 64.2 66.159.5 37.7 PIP-45Cc-1 — — — 92.9 93.6 92.9 63.1 65.6 59.5 38.1 PIP-45Cd-1— — — — 96.2 93.8 64.4 65.4 59.4 37.9 PIP-45Ce-1 — — — — — 94.6 64.465.9 59.8 37.6 PIP-45Cf-1 — — — — — — 65.2 67.0 61.0 38.8 PIP-45Da-1 — —— — — — — 66.6 59.9 37.6 PIP-45Db-1 — — — — — — — — 66.2 37.0 PIP-45Ea-135.7

Table 8 shows the percent sequence identity between the PIP-45-2polypeptide homologs. FIG. 2a-2l shows an amino acid sequence alignmentof the PIP-45-2 polypeptide homologs.

TABLE 8 PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- 45Ab-2 45Ac-245Ad-2 45Ae-2 45Af-2 45Ba-2 45Bb-2 45Bc-2 45Bd-2 45Be-2 SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 4 NO: 6 NO:8 NO: 10 NO: 12 NO: 14 NO: 16 NO: 18 NO: 20 NO: 22 PIP-45Aa-2 98.7 96.394.4 98.7 99.1 84.0 83.9 83.4 84.3 78.0 PIP-45Ab-2 — 96.1 94.2 99.6 98.984.2 84.1 83.6 84.1 78.2 PIP-45Ac-2 — — 95.7 96.1 96.4 84.3 84.3 83.884.1 79.1 PIP-45Ad-2 — — — 94.6 94.6 84.0 83.7 83.6 83.4 79.5 PIP-45Ae-2— — — — 98.9 84.2 84.1 83.6 84.1 78.2 PIP-45Af-2 — — — — — 84.1 84.183.6 84.7 78.2 PIP-45Ba-2 — — — — — — 99.3 98.3 95.5 76.2 PIP-45Bb-2 — —— — — — — 98.3 95.9 75.9 PIP-45Bc-2 — — — — — — — — 95.0 75.6 PIP-45Bd-2— — — — — — — — — 76.3 PIP-45Be-2 — — — — — — — — — — PIP-45Bf-2 — — — —— — — — — — PIP-45Bg-2 — — — — — — — — — — PIP-45Bh-2 — — — — — — — — —— PIP-45Bi-2 — — — — — — — — — — PIP-45Bj-2 — — — — — — — — — —PIP-45Bk-2 — — — — — — — — — — PIP-45Bl-2 — — — — — — — — — — PIP-45Bm-2— — — — — — — — — — PIP-45Ca-2 — — — — — — — — — — PIP-45Cb-2 — — — — —— — — — — PIP-45Cc-2 — — — — — — — — — — PIP-45Cd-2 — — — — — — — — — —PIP-45Ce-2 — — — — — — — — — — PIP-45Cf-2 — — — — — — — — — — PIP-45Da-2— — — — — — — — — — PIP-45Db-2 — — — — — — — — — — PIP-45Ea-2 — — — — —— — — — — PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- 45Bf-2 45Bg-245Bh-2 45Bi-2 45Bj-2 45Bk-2 45Bl-2 45Bm-2 45Ca-2 SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 24 NO: 26 NO: 28 NO: 30NO: 32 NO: 34 NO: 233 NO: 235 NO: 36 PIP-45Aa-2 80.1 83.6 83.9 83.4 83.083.7 82.4 82.6 67.0 PIP-45Ab-2 79.9 83.8 84.1 83.6 83.0 83.6 82.8 82.666.9 PIP-45Ac-2 79.7 83.9 84.3 83.7 83.7 83.6 82.8 82.6 67.7 PIP-45Ad-280.8 83.4 83.9 83.6 83.6 83.9 82.4 82.1 67.3 PIP-45Ae-2 79.9 83.8 84.183.6 83.0 83.6 82.6 82.5 66.9 PIP-45Af-2 79.9 83.7 84.1 83.6 83.4 83.982.6 82.8 67.2 PIP-45Ba-2 76.7 98.9 99.3 97.9 91.2 90.1 91.6 90.1 67.2PIP-45Bb-2 76.5 99.3 99.3 98.3 91.4 90.1 91.6 90.1 67.3 PIP-45Bc-2 76.497.9 98.3 99.6 90.8 89.2 90.7 89.9 67.3 PIP-45Bd-2 76.7 95.5 95.7 94.991.8 89.7 91.8 90.1 67.2 PIP-45Be-2 88.7 75.6 76.1 75.6 75.9 75.9 76.575.7 67.9 PIP-45Bf-2 — 76.1 76.7 76.3 77.2 77.4 77.1 76.3 68.7PIP-45Bg-2 — — 98.9 98.3 91.0 89.7 91.2 89.7 67.3 PIP-45Bh-2 — — — 98.391.4 90.3 91.8 90.3 67.2 PIP-45Bi-2 — — — — 90.8 89.1 90.6 89.9 67.3PIP-45Bj-2 — — — — — 88.2 88.8 87.5 68.3 PIP-45Bk-2 — — — — — — 89.086.5 66.6 PIP-45Bl-2 — — — — — — — 88.2 68.3 PIP-45Bm-2 — — — — — — — —66.8 PIP-45Ca-2 — — — — — — — — — PIP-45Cb-2 — — — — — — — — —PIP-45Cc-2 — — — — — — — — — PIP-45Cd-2 — — — — — — — — — PIP-45Ce-2 — —— — — — — — — PIP-45Cf-2 — — — — — — — — — PIP-45Da-2 — — — — — — — — —PIP-45Db-2 — — — — — — — — — PIP-45Ea-2 — — — — — — — — — PIP- PIP- PIP-PIP- PIP- PIP- PIP- PIP- PIP- 45Cb-2 45Cc-2 45Cd-2 45Ce-2 45Cf-2 45Da-245Db-2 45Ea-2 45Ga-2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID NO: 38 NO: 40 NO: 42 NO: 44 NO: 237 NO: 46 NO: 48 NO: 50NO: 52 PIP-45Aa-2 66.8 68.6 67.7 68.3 66.8 56.6 51.3 47.4 30.6PIP-45Ab-2 66.9 68.5 67.6 68.1 66.7 56.5 51.4 47.7 30.1 PIP-45Ac-2 67.568.8 68.1 68.1 67.3 56.4 51.8 47.7 30.8 PIP-45Ad-2 67.5 68.8 67.9 68.167.0 57.3 52.6 48.4 29.8 PIP-45Ae-2 66.7 68.5 67.8 68.1 66.7 56.5 51.447.7 30.1 PIP-45Af-2 66.8 68.6 67.9 68.5 67.0 56.4 51.3 47.8 30.6PIP-45Ba-2 67.4 67.9 67.3 67.9 67.1 58.1 52.6 49.3 30.5 PIP-45Bb-2 67.968.3 67.5 68.0 67.2 57.8 52.4 49.5 30.2 PIP-45Bc-2 67.9 68.3 67.5 68.067.2 58.0 52.3 49.8 30.8 PIP-45Bd-2 68.1 67.9 67.3 67.7 67.2 57.6 52.650.2 31.1 PIP-45Be-2 67.7 70.2 67.7 68.9 67.7 58.8 51.9 48.3 30.8PIP-45Bf-2 69.1 69.9 69.7 70.2 68.8 58.6 53.4 47.9 32.1 PIP-45Bg-2 68.168.3 67.7 68.1 67.2 57.6 52.5 49.5 30.4 PIP-45Bh-2 67.4 67.9 67.2 67.766.7 58.3 52.5 49.7 30.5 PIP-45Bi-2 67.9 68.3 67.5 68.0 67.2 58.0 52.649.9 30.9 PIP-45Bj-2 69.0 68.5 68.3 68.5 67.9 56.7 52.0 48.0 31.8PIP-45Bk-2 67.3 67.5 66.8 67.0 66.3 57.8 53.5 49.4 31.2 PIP-45Bl-2 68.568.6 67.3 67.9 67.5 56.7 53.4 49.4 31.5 PIP-45Bm-2 66.2 67.3 67.0 67.566.7 57.0 51.8 48.7 31.9 PIP-45Ca-2 93.0 89.0 92.1 93.6 96.6 56.8 53.646.4 30.1 PIP-45Cb-2 — 89.4 91.7 93.8 93.2 56.6 53.0 46.5 31.5PIP-45Cc-2 — — 88.3 90.3 89.8 58.3 53.1 46.2 31.8 PIP-45Cd-2 — — — 93.692.6 56.5 51.9 46.1 31.6 PIP-45Ce-2 — — — — 94.7 56.9 53.6 46.7 32.5PIP-45Cf-2 — — — — — 57.2 53.7 47.0 32.1 PIP-45Da-2 — — — — — — 55.648.6 32.0 PIP-45Db-2 — — — — — — — 49.1 30.4 PIP-45Ea-2 — — — — — — — —32.5

Table 9 shows the PIP-64-1 polypeptide and PIP-64-2 polypeptide homologsidentified, sequence identification numbers for each and the bacterialstrains they were identified from. Table 10 shows the percent sequenceidentity between the PIP-64-1 polypeptide homologs. FIG. 3a-3b shows anamino acid sequence alignment of the PIP-64-1 polypeptide homologs.Table 11 shows the percent sequence identity between the PIP-64-2polypeptide homologs. FIG. 4a-4b shows an amino acid sequence alignmentof the PIP-64-2 polypeptide homologs.

TABLE 9 Gene Source Species PIP-64Aa-1 SEQ ID NO: 53 LBV9691 Pseudomonasbrenneri PIP-64Aa-2 SEQ ID NO: 54 PIP-64Aa-1 SEQ ID NO: 53 LBV10925;LBV10914 Pseudomonas gessardii PIP-64Ab-2 SEQ ID NO: 55 PIP-64Ba-1 SEQID NO: 238 internal collection - SSP560F2b Pseudomonas entomophilaPIP-64Ba-2 SEQ ID NO: 239 PIP-64Ca-1 SEQ ID NO: 56 NCBI hypotheticalprotein WP_016977798 Pseudomonas fluorescens PIP-64Ca-2 SEQ ID NO: 57NCBI hypothetical protein WP_016977799 PIP-64Ea-1 SEQ ID NO: 58 internalDuPont collection P4G7 Alcaligenes faecalis PIP-64Ea-2 SEQ ID NO: 59PIP-64Eb-1 SEQ ID NO: 60 ATCC33950-internal genome sequence Alcaligenesfaecalis PIP-64Eb-2 SEQ ID NO: 61 PIP-64Ec-1 SEQ ID NO: 62EMBL-Uncharacterized protein Alcaligenes sp. HPC1271 PIP-64Ec-2 SEQ IDNO: 63 M5J334_9BURK and M5IW68_9BURK PIP-64Ga-1 SEQ ID NO: 64 EMBLR9VGC3_9ENTR Enterobacter PIP-64Ha-1 SEQ ID NO: 65 NCBI hypotheticalprotein PSF113_0646 Pseudomonas fluorescens PIP-64Ha-2 SEQ ID NO: 66(YP_005206077.1) and PSF113_0647 (YP_005206078.1) PIP-64Hb-1 SEQ ID NO:67 NCBI-hypothetical protein PSEBR_a622 Pseudomonas brassicacearumPIP-64Hb-2 SEQ ID NO: 68 (YP_004351774.1) and PSEBR_a623(YP_004351775.1) PIP-64Hc-1 SEQ ID NO: 69 JGI- SwiRh_808460 hypotheticalprotein Switchgrass rhizosphere PIP-64Hc-2 SEQ ID NO: 70 JGI-SwBS_00338360 hypothetical microbial community from protein MichiganPIP-64Hd-1 SEQ ID NO: 71 JGI- SwiRh_668170 hypothetical proteinMiscanthus rhizosphere PIP-64Hd-2 SEQ ID NO: 72 JGI - MRS2a_00580520hypothetical microbial communities from protein Kellogg

TABLE 10 PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- 64Ba-1 64Ca-164Ea-1 64Eb-1 64Ec-1 64Ga-1 64Ha-1 64Hb-1 64Hc-1 64Hd-1 SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 238 NO: 56NO: 58 NO: 60 NO: 62 NO: 64 NO: 65 NO: 67 NO: 69 NO: 71 PIP-64Aa-1 84.072.3 59.1 59.1 59.1 33.2 27.8 27.4 28.6 27.9 PIP-64Ba-1 — 74.2 56.4 56.856.4 31.4 25.7 26.9 26.0 27.2 PIP-64Ca-1 — — 55.6 55.6 55.6 32.1 23.723.4 25.2 26.4 PIP-64Ea-1 — — — 99.2 99.6 31.6 25.4 24.6 25.3 26.0PIP-64Eb-1 — — — — 98.8 31.6 25.0 24.6 25.3 26.0 PIP-64Ec-1 — — — — —31.6 25.4 24.6 25.3 26.0 PIP-64Ga-1 — — — — — — 22.1 23.1 22.4 22.6PIP-64Ha-1 — — — — — — — 94.6 66.2 62.1 PIP-64Hb-1 — — — — — — — — 66.262.1 PIP-64Hc-1 — — — — — — — — — 76.8

TABLE 11 PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- 64Ab-2 64Ba-264Ca-2 64Ea-2 64Eb-2 64Ec-2 64Ha-2 64Hb-2 64Hc-2 64Hd-2 SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 55 NO: 239NO: 57 NO: 59 NO: 61 NO: 63 NO: 66 NO: 68 NO: 70 NO: 72 PIP-64Aa-2 95.866.9 54.8 38.9 40.0 39.6 24.0 24.8 24.0 23.0 PIP-64Ab-2 — 66.2 54.8 39.641.2 40.8 25.9 23.1 22.3 22.3 PIP-64Ba-2 — — 49.8 38.5 41.1 39.6 21.324.4 24.0 19.0 PIP-64Ca-2 — — — 39.0 42.5 40.6 22.5 24.5 23.8 22.5PIP-64Ea-2 — — — — 85.4 90.7 22.3 18.2 22.0 21.9 PIP-64Eb-2 — — — — —93.4 25.5 21.6 22.7 21.9 PIP-64Ec-2 — — — — — — 23.3 19.0 23.0 21.7PIP-64Ha-2 — — — — — — — 72.3 49.2 35.7 PIP-64Hb-2 — — — — — — — — 52.538.5 PIP-64Hc-2 — — — — — — — — — 41.2

Table 12 shows the PIP-74-1 polypeptide and PIP-74-2 polypeptidehomologs identified, sequence identification numbers for each and thebacterial strains they were identified from. Table 13 shows the percentsequence identity between the PIP-74-1 polypeptide family members. FIG.5a-5b shows an amino acid sequence alignment of the PIP-74-1 polypeptidehomologs. Table 14 shows the percent sequence identity between thePIP-74-2 polypeptide family members. FIG. 6 shows an amino acid sequencealignment of the PIP-74-2 polypeptide homologs.

TABLE 12 Gene Source Species PIP-74Aa-1 SEQ ID NO: 73 internalcollection - Pseudomonas PIP-74Aa-2 SEQ ID NO: 74 SS135B4b rhodesiaePIP-74Ab-1 SEQ ID NO: 75 internal collection - Pseudomonas PIP-74Ab-2SEQ ID NO: 76 SSP427D6-1 orientalis PIP-74Ca-1 SEQ ID NO: 77 internalcollection - Pseudomonas PIP-74Ca-2 SEQ ID NO: 78 JH21146-1 sp. PKRS11

TABLE 13 PIP-74Ab-1 PIP-74Ca-1 PIP-74Aa-1 99.6 74.5 PIP-74Ab-1 — 74.5

TABLE 14 PIP-74Ab-2 PIP-74Ca-2 PIP-74Aa-2 98.0 66.3 PIP-74Ab-2 — 66.3

Table 15 shows the PIP-75 polypeptide homologs identified, sequenceidentification numbers for each and the bacterial strains they wereidentified from. Table 16 shows the percent sequence identity betweenthe PIP-75 polypeptide family members. FIG. 7 shows an amino acidsequence alignment of the PIP-75 polypeptide homologs.

TABLE 15 Gene Source Species PIP-75Aa SEQ ID NO: 79 LBV6019 Pseudomonasantarctica PIP-75Ba SEQ ID NO: 80 LBV2669 Pseudomonas orientalisPIP-75Da SEQ ID NO: 81 internal - JH34920-1 Enterobacter asburiaePIP-75Ea SEQ ID NO: 82 NCBI A936_14984 Enterobacter sp. PIP-75Ga SEQ IDNO: 83 NCBI-YP_004234966 Acidovorax avenae subsp. avenae ATCC 19860PIP-75Gb SEQ ID NO: 84 internal collection - SSP443E10-1 Serratiaplymuthica PIP-75Gc SEQ ID NO: 85 internal collection - JH20785-4Serratia liquefaciens PIP-75Gd SEQ ID NO: 86 internal collection -SSP291H3-2 Serratia sp. PIP-75Ge SEQ ID NO: 87 internal collection -JH20487-2 Serratia sp.

TABLE 16 PIP-75Ba PIP-75Da PIP-75Ea PIP-75Ga PIP-75Gb PIP-75Gc PIP-75GdPIP-75Ge PIP-75Aa 83.3 65.6 60.4 33.6 36.7 33.7 33.7 32.7 PIP-75Ba —65.6 59.4 33.6 34.0 32.0 38.1 31.6 PIP-75Da — — 85.3 35.2 39.6 38.5 32.334.4 PIP-75Ea — — — 35.8 37.5 35.4 30.2 31.2 PIP-75Ga — — — — 24.8 25.224.6 23.8 PIP-75Gb — — — — — 86.2 67.0 75.5 PIP-75Gc — — — — — — 61.373.1 PIP-75Gd — — — — — — — 82.1

Table 17 shows the PIP-77 polypeptide homologs identified, sequenceidentification numbers for each and the bacterial strains they wereidentified from. Table 18 shows the percent sequence identity betweenthe PIP-77 polypeptide family members. FIG. 8a-8b shows an amino acidsequence alignment of the PIP-77 polypeptide homologs.

TABLE 17 Gene Source Species PIP-77Aa SEQ ID NO: 88 SSP344E5 and other29 internal strains Pseudomonas chlororaphis PIP-77Ab SEQ ID NO: 89SSP346A11a Pseudomonas chlororaphis PIP-77Ac SEQ ID NO: 90 JH19897-4;JH19820-2; JH19887-2; JH20257- Pseudomonas 4; JH19881-4; JH19896-4;JH20401-2; brassicacearum PIP-77Ad SEQ ID NO: 91 SSP423G5-1; SSP344E7a;SSP283E1-2; Pseudomonas chlororaphis SSP283E2-1; SSP283E6-1; SSP259H3-2;JH20450-1; SSP459A9-4; SSP459B9-3; JH21227-2; JH22700-1; NCBI-WP_007925627; PIP-77Ae SEQ ID NO: 92 SSP346D1a Pseudomonas chlororaphisPIP-77Af SEQ ID NO: 240 NCBI hypothetical protein WP_023965133.1;Pseudomonas chlororaphis internal collection- SSP555A5b PIP-77Ba SEQ IDNO: 93 JH17731-2; JH17330-1; JH17729-3; JH17728- Pseudomonas fluorescens1; JH17574-1; JH17564-4 PIP-77Bb SEQ ID NO: 94 JH20704-3; JH20495-2Pseudomonas fluorescens PIP-77Bc SEQ ID NO: 95 JH18994-3; JH18447-2Pseudomonas fluorescens PIP-77Bd SEQ ID NO: 96 JH17494-4; JH17581-1;JH19353-3; JH17541- Pseudomonas fluorescens 1; JH17554-4; JH16392-2;JH17546-4; JH17696-1; JH17549-1; JH17110-1; SSP454G12-1; JH17430-2PIP-77Be SEQ ID NO: 97 SSP347B8a Pseudomonas fluorescens PIP-77Bf SEQ IDNO: 98 JH18110-4; JH18354-4; JH18107-3; Pseudomonas rhodesiae SSP450C9-1PIP-77Bg SEQ ID NO: 99 NCBI-WP_007969132 Pseudomonas-sp PIP-77Bh SEQ IDNO: 241 internal collection - SSP535F3b Pseudomonas rhodesiae PIP-77BiSEQ ID NO: 242 internal collection - SSP557G7-4 Pseudomonas rhodesiaePIP-77Ca SEQ ID NO: 100 SS154F1; SSP154F5a Pseudomonas fluorescensPIP-77Ea SEQ ID NO: 101 NCBI-WP_008458969 Enterobacter-sp PIP-77Eb SEQID NO: 102 NCBI-YP_564720 Shewanella denitrificans PIP-77Ec SEQ ID NO:103 NCBI-WP_005351930 Aeromonas diversa PIP-77Ed SEQ ID NO: 104NCBI-YP_001141694 Aeromonas salmonicida PIP-77Ee SEQ ID NO: 105NCBI-YP_004392889 Aeromonas veronii PIP-77Ef SEQ ID NO: 106NCBI-WP_005909090 Aeromonas molluscorum PIP-77Eq SEQ ID NO: 107NCBI-WP_010633780 Aeromonas aquariorum PIP-77Eh SEQ ID NO: 243 NCBIhypothetical protein Aeromonas hydrophila AH4AK4_1885 AHE49340 PIP-77EiSEQ ID NO: 244 EMBL-U1H356_9GAMM Aeromonas veronii PIP-77Ej SEQ ID NO:245 internal collection - JH58766-1 Haemophilus piscium

TABLE 18 PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP-PIP- 77Ab 77Ac 77Ad 77Ae 77Af 77Ba 77Bb 77Bc 77Bd 77Be 77Bf 77Bg 77BhSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO:89 90 91 92 240 93 94 95 96 97 98 99 241 PIP-77Aa 94.4 93.3 92.1 92.192.1 87.6 86.5 85.4 84.3 84.3 83.1 85.4 86.5 PIP-77Ab — 95.5 96.6 94.495.5 86.5 85.4 84.3 83.1 83.1 82.0 84.3 85.4 PIP-77Ac — — 98.9 95.5 98.985.4 84.3 83.1 82.0 83.1 80.9 82.0 84.3 PIP-77Ad — — — 96.6 98.9 86.585.4 84.3 83.1 84.3 82.0 82.0 85.4 PIP-77Ae — — — — 95.5 85.4 84.3 83.182.0 84.3 80.9 84.3 84.3 PIP-77Af — — — — — 85.4 84.3 83.1 82.0 83.180.9 82.0 84.3 PIP-77Ba — — — — — — 97.8 96.6 95.5 88.8 94.4 80.9 98.9PIP-77Bb — — — — — — — 98.9 97.8 86.5 96.6 82.0 96.6 PIP-77Bc — — — — —— — — 96.6 85.4 95.5 80.9 95.5 PIP-77Bd — — — — — — — — — 84.3 98.9 79.894.4 PIP-77Be — — — — — — — — — — 83.1 78.7 89.9 PIP-77Bf — — — — — — —— — — — 78.7 93.3 PIP-77Bg — — — — — — — — — — — — 82.0 PIP-77Bh — — — —— — — — — — — — — PIP-77Bi — — — — — — — — — — — — — PIP-77Ca — — — — —— — — — — — — — PIP-77Ea — — — — — — — — — — — — — PIP-77Eb — — — — — —— — — — — — — PIP-77Ec — — — — — — — — — — — — — PIP-77Ed — — — — — — —— — — — — — PIP-77Ee — — — — — — — — — — — — — PIP-77Ef — — — — — — — —— — — — — PIP-77Eg — — — — — — — — — — — — — PIP-77Eh — — — — — — — — —— — — — PIP-77Ei — — — — — — — — — — — — —

TABLE 18 PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP- PIP-77Bi 77Ca 77Ea 77Eb 77Ec 77Ed 77Ee 77Ef 77Eg 77Eh 77Ei 77Ej SEQ ID SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDNO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: 242 100 101 102 103 104105 106 107 243 244 245 PIP-77Aa 84.3 78.0 56.5 55.1 55.6 55.6 56.7 51.156.7 56.7 55.6 55.6 PIP-77Ab 83.1 76.9 58.7 55.1 56.7 55.6 56.7 51.156.7 56.7 55.6 55.6 PIP-77Ac 80.9 79.1 60.9 56.2 55.6 54.4 55.6 50.055.6 55.6 54.4 54.4 PIP-77Ad 82.0 78.0 60.9 56.2 55.6 53.3 54.4 48.954.4 54.4 53.3 53.3 PIP-77Ae 80.9 78.0 58.7 55.1 54.4 52.2 53.3 47.853.3 53.3 52.2 52.2 PIP-77Af 80.9 78.0 60.9 56.2 55.6 53.3 54.4 48.954.4 54.4 53.3 53.3 PIP-77Ba 94.4 74.7 57.6 51.7 54.4 53.3 54.4 47.853.3 53.3 52.2 53.3 PIP-77Bb 96.6 75.8 57.6 51.7 55.6 54.4 55.6 48.954.4 54.4 53.3 54.4 PIP-77Bc 95.5 74.7 57.6 51.7 54.4 53.3 54.4 47.853.3 53.3 52.2 53.3 PIP-77Bd 98.9 73.6 55.4 49.4 53.3 52.2 53.3 48.952.2 52.2 51.1 52.2 PIP-77Be 83.1 78.0 57.6 53.9 51.1 48.9 50.0 45.651.1 51.1 48.9 48.9 PIP-77Bf 97.8 72.5 54.3 49.4 52.2 52.2 53.3 48.952.2 52.2 51.1 52.2 PIP-77Bg 79.8 75.8 56.5 55.1 56.7 55.6 56.7 51.156.7 56.7 55.6 55.6 PIP-77Bh 93.3 75.8 58.7 52.8 53.3 52.2 53.3 46.752.2 52.2 51.1 52.2 PIP-77Bi — 72.5 55.4 49.4 53.3 52.2 53.3 48.9 52.252.2 51.1 52.2 PIP-77Ca — — 62.4 57.1 57.6 54.3 55.4 51.1 56.5 56.5 54.354.3 PIP-77Ea — — — 57.6 53.8 54.8 54.8 50.0 55.9 55.9 53.8 55.9PIP-77Eb — — — — 53.3 51.1 51.1 46.7 53.3 53.3 53.3 51.1 PIP-77Ec — — —— — 86.7 85.6 70.3 85.6 84.4 84.4 86.7 PIP-77Ed — — — — — — 96.7 75.888.9 90.0 94.4 90.0 PIP-77Ee — — — — — — — 75.6 87.8 88.9 97.8 88.9PIP-77Ef — — — — — — — — 84.6 85.7 75.6 80.2 PIP-77Eg — — — — — — — — —98.9 87.8 95.6 PIP-77Eh — — — — — — — — — — 88.9 94.4 PIP-77Ei — — — — —— — — — — — 86.7

Example 7 Functional Test of PIP-45-1 and PIP-45-2 Components fromDifferent Origins

In order to test the functionality of the PIP-45-1 and PIP-45-2components from different PIP-45 homologs, five selected active homologpairs were expressed individually (listed in Table 19). EachPIP-45-1component was mixed with every one of the five PIP-45-2components. All of the pairs were tested for WCRW insecticidal activityin diet based assays as described above. The results are shown in Table19, indicating that when paired together PIP-45-1 and PIP-45-2components from different sources can provide insecticidal activity.

TABLE 19 PIP045Aa-2 PIP045Ad-2 PIP045Ba-2 PIP045Ca-2 PIP045Cb-2 ActivitySEQ ID NO: 2 SEQ ID NO: 8 SEQ ID NO: 14 SEQ ID NO: 36 SEQ ID NO: 38PIP045Aa-1 Active Active Active Active Active SEQ ID NO: 1 PIP045Ad-1Active Active Active Active Active SEQ ID NO: 7 PIP045Ba-1 ActiveInactive Active Inactive Inactive SEQ ID NO: 13 PIP045Ca-1 InactiveInactive Inactive Active Active SEQ ID NO: 35 PIP045Cb-1 InactiveInactive Inactive Active Active SEQ ID NO: 37 Active in bold = from theoriginal pair; Active = insecticidal activity detected with componentsfrom different origins

Example 8 Gene Subcloning and E. Coli Expression

The target genes encoding the insecticidal proteins were first amplifiedby PCR using their genomic DNA as templates. The PCR primers weredesigned based on the 5′ end an3′ end sequences with appropriaterestriction sites incorporated. After restriction enzyme digestion, thePCR products were cloned into various E. coli expression vectors, i.e.pCOLD™ 1 with N-His tag, pMAL™ vector with and without MBP fusion orpCOLD™ vectors with and without His tag. The proteins were expressed inBL21(DE3) or C41 E. coli hosts cells with 1 mM IPTG overnight inductionat 16° C.

The recombinant protein was extracted from E. coli culture afterinduction, purified and assayed on insect targets as described inExample 1.

Example 9 Transient Expression and Insect Bioassay on Transient LeafTissues

Polynucleotides encoding both PIP-64Aa-1 (SEQ ID NO: 160) and PIP-64Aa-2(SEQ ID NO: 161) were cloned into a transient expression vector undercontrol of a viral promoter pDMMV respectively (Day, et. al., (1999)Plant Mol. Biol. 40:771-782). The agro-infiltration method ofintroducing an Agrobacterium cell suspension to plant cells of intacttissues so that reproducible infection and subsequent plant derivedtransgene expression may be measured or studied is well known in the art(Kapila, et. al., (1997) Plant Science 122:101-108). Briefly, youngplantlets of bush bean (common bean, Phaseolus vulgaris) and soybean(Glycine max), were agro-infiltrated with normalized bacterial cellcultures of test and control strains. 10 leaf discs were generated foreach plantlets and infested with 3 neonates of both Soy Bean Looper(SBL) (Pseudoplusia includes) and Velvet bean caterpillar (VBC) (VelvetAnticarsia gemmatalis) alone with two controls of leaf discs generatedwith Agrobacterium only and DsRed2 fluorescence marker (Clontech,Mountain View, Calif.) expression vector in Agrobacterium. Theconsumption of green leaf tissues was scored two days after infestation.Transient protein expressions of both PIP-64-1 (SEQ ID NO: 53) andPIP-64-2 (SEQ ID NO: 54) were confirmed by Mass spectrometry basedprotein identification method using extracted protein lysates fromco-infiltrated leaf tissues (Patterson, (1998) 10(22):1-24, CurrentProtocol in Molecular Biology published by John Wiley & Son Inc). Thetransiently co-expressed PIP-64-1 (SEQ ID NO: 53) and PIP-64-2 (SEQ IDNO: 54) protected leaf discs from consumption by the infested insectswhile total green tissue consumption was observed for the two negativecontrols.

Example 10 Agrobacterium-Mediated Stable Transformation of Maize

For Agrobacterium-mediated maize transformation of insecticidalpolypeptides, the method of Zhao is employed (U.S. Pat. No. 5,981,840and International Patent Publication Number WO 1998/32326, the contentsof which are hereby incorporated by reference). Briefly, immatureembryos are isolated from maize and the embryos contacted with anAgrobacterium suspension, where the bacteria were capable oftransferring a polynucleotide encoding an insecticidal polypeptide ofthe disclosure to at least one cell of at least one of the immatureembryos (step 1: the infection step). In this step the immature embryosare immersed in an Agrobacterium suspension for the initiation ofinoculation. The embryos are co-cultured for a time with theAgrobacterium (step 2: the co-cultivation step). The immature embryosare cultured on solid medium with antibiotic, but without a selectingagent, for Agrobacterium elimination and for a resting phase for theinfected cells. Next, inoculated embryos are cultured on mediumcontaining a selective agent and growing transformed callus is recovered(step 4: the selection step). The immature embryos are cultured on solidmedium with a selective agent resulting in the selective growth oftransformed cells. The callus is then regenerated into plants (step 5:the regeneration step), and calli grown on selective medium are culturedon solid medium to regenerate the plants.

For detection of the insecticidal polypeptide in leaf tissue 4lyophilized leaf punches/sample are pulverized and resuspended in 100 μLPBS containing 0.1% TWEEN™ 20 (PBST), 1% beta-mercaoptoethanolcontaining 1 tablet/7 mL complete Mini proteinase inhibitor (Roche1183615301). The suspension is sonicated for 2 min and then centrifugedat 4° C., 20,000 g for 15 min. To a supernatant aliquot 1/3 volume of 3×NuPAGE® LDS Sample Buffer (Invitrogen™ (CA, USA), 1% B-ME containing 1tablet/7 mL complete Mini proteinase inhibitor was added. The reactionis heated at 80° C. for 10 min and then centrifuged. A supernatantsample is loaded on 4-12% Bis-Tris Midi gels with MES running buffer asper manufacturer's (Invitrogen™) instructions and transferred onto anitrocellulose membrane using an iBlot® apparatus (Invitrogen™). Thenitrocellulose membrane is incubated in PBST containing 5% skim milkpowder for 2 hours before overnight incubation in affinity-purifiedrabbit anti-insecticidal polypeptide in PBST overnight. The membrane isrinsed three times with PBST and then incubated in PBST for 15 min andthen two times 5 min before incubating for 2 hours in PBST with goatanti-rabbit-HRP for 3 hours. The detected proteins are visualized usingECL Western Blotting Reagents (GE Healthcare cat #RPN2106) and Kodak®Biomax® MR film. For detection of the insecticidal protein in roots theroots are lyophilized and 2 mg powder per sample is resuspended in LDS,1% beta-mercaptoethanol containing 1 tablet/7 mL Complete Miniproteinase inhibitor is added. The reaction is heated at 80° C. for 10min and then centrifuged at 4° C., 20,000g for 15 min. A supernatantsample is loaded on 4-12% Bis-Tris Midi gels with MES running buffer asper manufacturer's (Invitrogen™) instructions and transferred onto anitrocellulose membrane using an iBlot® apparatus (Invitrogen™). Thenitrocellulose membrane is incubated in PBST containing 5% skim milkpowder for 2 hours before overnight incubation in affinity-purifiedpolyclonal rabbit anti-insecticidal antibody in PBST overnight. Themembrane is rinsed three times with PBST and then incubated in PBST for15 min and then two times 5 min before incubating for 2 hours in PBSTwith goat anti-rabbit-HRP for 3 hrs. The antibody bound insecticidalproteins are detected using ECL™ Western Blotting Reagents (GEHealthcare cat #RPN2106) and Kodak® Biomax® MR film.

Transgenic maize plants positive for expression of the insecticidalproteins are tested for pesticidal activity using standard bioassaysknown in the art. Such methods include, for example, root excisionbioassays and whole plant bioassays. See, e.g., US Patent ApplicationPublication Number US 2003/0120054 and International Publication NumberWO 2003/018810.

Example 11 Expression Vector Constructs for Expression of InsecticidalPolypeptides in Plants

The plant expression vectors, can be constructed to include a transgenecassette containing the coding sequence pf the insecticidal polypeptide,under control of the Mirabilis Mosaic Virus (MMV) promoter [Dey N andMaiti I B, 1999, Plant Mol. Biol. 40(5):771-82] in combination with anenhancer element. These constructs can be used to generate transgenicmaize events to test for efficacy against corn rootworm provided byexpression of the insecticidal polypeptide of the disclosure.

T0 greenhouse efficacy of the events can be measured by root protectionfrom Western corn rootworm. Root protection is measured according to thenumber of nodes of roots injured (CRWNIS=corn rootworm node injuryscore) using the method developed by Oleson, et al. (2005) [J. EconEntomol. 98(1):1-8]. The root injury score is measured from “0” to “3”with “0” indicating no visible root injury, “1” indicating 1 node ofroot damage, “2” indicating 2 nodes or root damage, and “3” indicating amaximum score of 3 nodes of root damage. Intermediate scores (e.g. 1.5)indicate additional fractions of nodes of damage (e.g. one and a halfnodes injured).

That which is claimed:
 1. An insecticidal polypeptide selected from: a)a PIP-45-1 polypeptide comprising an amino acid sequence having greaterthan 80% sequence identity compared to the amino acid sequence of SEQ IDNO: 1; b) a PIP-45-1 polypeptide comprising an amino acid sequenceselected from SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21,SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45, SEQ IDNO: 234 and SEQ ID NO: 236; c) a PIP-45-2 polypeptide comprising anamino acid sequence having greater than 80% sequence identity comparedto the amino acid sequence of SEQ ID NO: 2; d) a PIP-45-2 polypeptidecomprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 18, SEQ IDNO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44,SEQ ID NO: 46, SEQ ID NO: 235 or SEQ ID NO: 237, wherein the PIP-45-1polypeptide of a) or b) and the PIP-45-2 polypeptide of c) or d) incombination have insecticidal activity against Western corn rootworm. 2.An insecticidal composition comprising the PIP-45-1 polypeptide of claim1a) or 1b) and the PIP-45-2 polypeptide of claim 1c) or 1d). 3.(canceled)
 4. (canceled)
 5. (canceled)
 6. (canceled)
 7. (canceled)
 8. Arecombinant polynucleotide encoding an insecticidal polypeptide selectedfrom: a) a PIP-45-1 polypeptide comprising an amino acid sequence havinggreater than 80% sequence identity compared to the amino acid sequenceof SEQ ID NO: 1; b) a PIP-45-1 polypeptide comprising an amino acidsequence selected from SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31,SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO:45, SEQ ID NO: 234 and SEQ ID NO: 236; c) a PIP-45-2 polypeptidecomprising an amino acid sequence having greater than 80% sequenceidentity compared to the amino acid sequence of SEQ ID NO: 2; and d) aPIP-45-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 2,SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ IDNO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 235 or SEQ ID NO: 237;9. The recombinant polynucleotide of claim 8, wherein the recombinantpolynucleotide is selected from: a) the polynucleotide of SEQ ID NO:108, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO:142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 220 orSEQ ID NO: 222; and b) the polynucleotide of SEQ ID NO: 109, SEQ ID NO:125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 135, SEQID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:147, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 221 or SEQ ID NO: 223.10. A DNA construct comprising, i) the recombinant polynucleotide ofclaim 8a), 8b) or 9a) encoding the PIP-45-1 polypeptide; ii) and/or therecombinant polynucleotide of claim 8c), 8d) or 9b) encoding thePIP-45-2 polypeptide; and iii) at least one heterologous regulatorysequence operably linked to the recombinant polynucleotides.
 11. Atransgenic plant or plant cell comprising the DNA construct of claim 10.12. A method of inhibiting growth or killing an agricultural insect pestpopulation, comprising contacting the insect pest population with aninsecticidally-effective amount of a) a PIP-45-1 polypeptide selectedfrom: i. a PIP-45 polypeptide comprising an amino acid sequence havinggreater than 80% sequence identity compared to the amino acid sequenceof SEQ ID NO: 1; and ii. a PIP-45-1 polypeptide comprising an amino acidsequence selected from SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31,SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO:45, SEQ ID NO: 234 and SEQ ID NO: 236; and b) a PIP-45-2 polypeptideselected from: i. a PIP-45-2 polypeptide comprising an amino acidsequence having greater than 80% sequence identity compared to the aminoacid sequence of SEQ ID NO: 2; and ii. a PIP-45-2 polypeptide comprisesthe amino acid sequence of SEQ ID NO: 2, SEQ-ID NO: 18, SEQ ID NO: 20,SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ IDNO: 46, SEQ ID NO: 235 or SEQ ID NO:
 237. 13. (canceled)
 14. (canceled)15. (canceled)
 16. (canceled)
 17. (canceled)
 18. A method of controllingLepidoptera and/or Coleoptera insect infestation in a transgenic plantand providing insect resistance management, comprising expressing in theplant; a) a polynucleotide encoding a PIP-45-1 polypeptide selectedfrom: i. a PIP-45 polypeptide comprising an amino acid sequence havinggreater than 80% sequence identity compared to the amino acid sequenceof SEQ ID NO: 1; and ii. a PIP-45-1 polypeptide comprising an amino acidsequence selected from SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31,SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO:45, SEQ ID NO: 234 and SEQ ID NO: 236; and b) a polynucleotide encodinga PIP-45-2 polypeptide selected from: i. a PIP-45-2 polypeptidecomprising an amino acid sequence having greater than 80% sequenceidentity compared to the amino acid sequence of SEQ ID NO: 2; and ii. aPIP-45-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 2,SEQ-ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ IDNO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 235 or SEQ ID NO: 237.19. The method of claim 12 or 18, wherein the insect pest or insect pestpopulation is resistant to a Bt toxin.
 20. (canceled)